Methods and apparatuses for sorting target particles

ABSTRACT

This disclosure provides methods and apparatuses for sorting target particles. In various embodiments, the disclosure provides a cassette for sorting target particles, methods for sorting target particles, methods of loading a microchannel for maintaining sample material viability, methods of quantifying sample material, and an optical apparatus for laser scanning and particle sorting.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/425,856, filed on May 29, 2019, now U.S. Pat. No. 10,722,885, whichis a continuation of U.S. patent application Ser. No. 16/044,166, filedon Jul. 24, 2018, now U.S. Pat. No. 10,350,595, which is a continuationof International Patent Application No. PCT/US2017/061414, filed on Nov.13, 2017, which claims priority to U.S. Provisional Application62/421,979, filed on Nov. 14, 2016, each of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Through conventional technologies, biological material may be screenedfor biological components, such as cells, antibodies, proteins,peptides, and nucleic acids. However, a number of challenges in theidentification, isolation and characterization of biological componentsstill remain. For example, some devices and methods require multiple,time-consuming selection steps. Additionally, some devices and methodsfail to prevent sample contamination, fail to accurately detect multiplepositive signals, fail to isolate viable cells, fail to detect cells,and fail to differentiate a single cell from multiple cells. Somedevices and methods are also limited in the number of cells that can bescreened with reasonable expediency.

Accordingly, there is a current need in the art for new methods andapparatuses for identifying, isolating, sorting and characterizingbiological material, in particular cellular material. The presentdisclosure addresses this deficiency with new methods and apparatusesfor sorting viable cellular material as well as other target particles.

SUMMARY OF THE INVENTION

The present disclosure provides new methods and apparatuses for sortingtarget particles, including viable cellular material, and the productsthereof.

In some aspects, the disclosure provides a cassette for detecting andsorting target particles, the cassette comprising a substrate with afirst surface and a second surface and a plurality of microchannelsextending from the first surface to the second surface, a first housingconfigured to receive the substrate wherein the first housing comprisesan internal surface to receive a target particle released from thesubstrate, a second housing coupled to the first housing in a mannerwherein the first and second housing together encapsulate the substrateand wherein the first or second housing further comprises a first fillport, and a transmissive portion located in one or each of the firsthousing and the second housing, wherein the transmissive portion permitstransmission of electromagnetic radiation from outside of the cassetteto the substrate.

In certain embodiments, the transmissive portion is at least partiallytransparent to a wavelength in the range of about 250 nm to 1600 nm. Incertain embodiments, the transmissive portion is located in the firsthousing. In some embodiments, the transmissive portion is located in thesecond housing.

In certain embodiments, the first fill port is configured to receive asample material mixture into the cassette. In certain embodiments, thefirst housing comprises the first fill port. In some embodiments, thesecond housing comprises the first fill port.

In certain embodiments, the first or second housing further comprises arelease port. In certain embodiments, the release port is in fluidcommunication with the internal surface to permit transfer of the targetparticles out of the release port in the cassette. In certainembodiments, the first housing comprises the release port. In someembodiments, the second housing comprises the release port.

In certain embodiments, the second housing is positioned on top of thefirst housing. In certain embodiments, the first housing is positionedin a substantially parallel plane relative to the second housing.

In some aspects, the disclosure provides a cassette for detecting andsorting target particles, the cassette comprising a substrate with afirst surface and a second surface and a plurality of microchannelsextending from the first surface to the second surface, a first housingconfigured to receive the substrate wherein the first housing comprisesan internal surface to receive a target particle released from thesubstrate, a second housing coupled to the first housing in a mannerwherein the first and second housing together encapsulate the substrate,and wherein the first or second housing further comprises a first fillport for introducing a target particle mixture into the cassette andwherein the first or second housing further comprises a release port forreleasing target particles from the cassette.

In certain embodiments, the first housing comprises the first fill port.In some embodiments, the second housing comprises the first fill port.

In certain embodiments, the first housing comprises the release port. Insome embodiments, the second housing comprises the release port.

In certain embodiments, the cassette further comprises a transmissiveportion located in one or each of the first housing and the secondhousing, wherein the transmissive portion permits transmission ofelectromagnetic radiation from outside of the cassette to the substrate.In certain embodiments, the transmissive portion is at least partiallytransparent to a wavelength in the range of about 250 nm to 1600 nm.

In certain embodiments, the second housing is positioned on top of thefirst housing. In certain embodiments, the first housing is positionedin a substantially parallel plane relative to the second housing. Invarious embodiments, the first housing and second housing are coupledirreversibly as a single housing unit. In some embodiments, the firstand second housing are coupled to one another in a reversible manner.

In certain embodiments, the substrate comprises glass. In certainembodiments, the plurality of microchannels is positioned substantiallyin parallel to each other. In certain embodiments, the plurality ofmicrochannels is from about 1 million to about 100 billionmicrochannels. In certain embodiments, the plurality of microchannelshas an average internal diameter of about 50 nm to about 500 sm. Incertain embodiments, the distance from the first surface to the secondsurface of the substrate is on average from about 10 μm to about 1 mm.In certain embodiments, the substrate further comprises border elementswhich extend vertically from the perimeter of the first surface of thesubstrate and permit containment of fluid on the first surface of thesubstrate.

In certain embodiments, the cassette further comprises a sample well influid communication with the first fill port, wherein the sample well isconfigured to load a sample material mixture into the microchannels ofthe substrate. The sample well may be in contact with the first surfaceof the substrate. The sample well may be movable across the firstsurface of the substrate. In certain embodiments, the sample well ismovable manually, mechanically, or electronically.

In certain embodiments, the first or second housing further comprise asecond fill port. The first or second fill port may be in fluidcommunication with the internal surface of the first housing. In certainembodiments, the first or second housing further comprise a third fillport. In certain embodiments, the cassette further comprises a hydrationmembrane positioned to contact the substrate or positioned adjacent tothe substrate. The first, second, or third fill port may be in fluidcommunication with the hydration membrane. In certain embodiments, thefirst and second housing prevent contaminant entry into the cassette. Incertain embodiments, the internal surface further comprises a collectionwell. The collection well may be in fluid communication with the releaseport.

In certain embodiments, the first or second housing further comprises ametal frame which is affixed to the first or second housing and thefirst or second surface of the substrate and applies tension across thesurface of the substrate. In certain embodiments, the substratecomprises a first end, a second end and a middle portion, wherein thefirst end, the second end and the middle portion are all substantiallywithin the same plane.

In certain embodiments, the target particles comprise cells. In certainembodiments, the cassette is sterilized before use.

In certain embodiments, the substrate has dimensions of 3 mm×3 mm×0.3 mmto 5000 mm×15000 mm×1000 mm. In certain embodiments, the substrate hasdimensions of 3 mm×3 mm×0.3 mm to 10000 mm×10000 mm×100 mm.

In certain embodiments, the cassette further comprises a contacttransducer in contact with one or more components of the cassette.

In some aspects, the disclosure provides a substrate comprising a firstsurface and a second surface and a plurality of microchannels extendingfrom the first surface to the second surface, wherein the microchannelscomprise target particles and opaque material wherein at least about 50%of the target particles are separated from the opaque material by atleast about 1 μm by a spacer comprising a transparent gel or transparentsolid or combination thereof. In certain embodiments, at least about 50%of the target particles are separated from the opaque material by atleast about 1 μm by a spacer comprising a transparent gel or transparentsolid, such as agarose, collagen, matrigel, alginate, and combinationsthereof.

In some aspects, the disclosure provides a kit comprising a cassette ofthe present disclosure and instructions for use thereof in the detectionand sorting of target particles.

In some aspects, the disclosure provides a method of detecting andsorting target particles, the method comprising adding a sample materialmixture into the cassette of the present disclosure, loading the samplematerial mixture into the microchannels of the substrate, scanning thecontents of the microchannels to detect microchannels containing one ormore target particles, and releasing the target particles from thesubstrate to the internal surface.

In certain embodiments, the sample material mixture is added into thecassette through the first fill port. The sample material mixture may beadded to the sample well. In some embodiments, the sample materialmixture is added to the substrate. In certain embodiments, the samplematerial mixture comprises a cellular suspension. The sample materialmixture comprises about 1×10⁶ to about 100×10⁹ cells. The sample wellmay load an approximately equivalent amount of the sample materialmixture in each microchannel of the substrate.

In certain embodiments, scanning the microchannels comprise illuminationof a microchannel with a first wavelength and detection of a secondwavelength from the microchannel, wherein the second wavelengthcorresponds with the target particles. Scanning the contents of themicrochannels may comprise illumination of a microchannel with aplurality of different wavelengths and detecting an emission from themicrochannel wherein the emission corresponds with one or more targetparticles. In certain embodiments, the scanning the contents of themicrochannels comprise illumination of a microchannel with a singlewavelength and detection of a plurality of emissions from themicrochannel, wherein the plurality of emissions correspond with one ormore target particles. In some embodiments, the scanning the contents ofthe microchannels comprises illumination of a microchannel with aplurality of different wavelengths and detection of a plurality ofemissions from the microchannel, wherein one or more of the emissionscorrespond with one or more target particles. In certain embodiments,the first and second wavelengths are independently selected from about200 nm to about 1.5 mm.

In certain embodiments, the target particle is released from thesubstrate to the internal surface with an energy from a thirdwavelength. In certain embodiments, the third wavelength is selectedfrom about 200 nm to about 1.5 mm, such as from about 350 nm to about1200 nm. In certain embodiments, the target particle is a cell.

In certain embodiments, following the release step, the method furthercomprises a step of transferring the target particle from the internalsurface to the release port. In certain embodiments, transferring thetarget particle from the internal surface to the release port comprisesadding a solution to the first or second fill port to transfer thetarget particle to the release port. The solution may be a buffersolution.

In certain embodiments, the method further comprises a step ofsonicating the substrate, wherein the sonication step occurs prior tothe scanning step. The sonication step may occur prior to, concurrentwith, or subsequent to the loading step or a combination thereof. Incertain embodiments, the sonication step comprises contacting thesubstrate with a contact transducer.

In some aspects, the disclosure provides a method of loading a mixtureinto a microchannel of substrate comprising a plurality ofmicrochannels, the method comprising adding a first mixture tomicrochannels of a substrate, wherein the first mixture comprises atransparent solution and a plurality of opaque particles, and adding asecond mixture to microchannels of a substrate, wherein the secondmixture comprises a sample component and an aqueous solution.

In certain embodiments, the transparent solution comprises a gellingagent, such as natural gums, starches, pectins, agar-agar, gelatin, or acombination thereof. In certain embodiments, the method furthercomprises allowing the first mixture to solidify prior to the additionof the second mixture. In certain embodiments, the method furthercomprises a step of adding a reagent to remove a portion of the firstmixture from the microchannels following the addition of the firstmixture to the microchannels and prior to addition of the second mixtureto the microchannels.

In certain embodiments, the sample component is a cell. In certainembodiments, the plurality of opaque particles absorb radiation at afourth wavelength. In certain embodiments, the fourth wavelength isselected from about 250 nm to about 1.5 mm. In certain embodiments, 90%or more of the plurality of opaque particles are not in contact with thesample component in the microchannels. In certain embodiments, theplurality of opaque particles are separated from the sample component bya distance of at least about 1 μm or more in the microchannels.

In some aspects, the disclosure provide a method of loading a mixtureinto a microchannel of substrate comprising a plurality ofmicrochannels, the method comprising adding a sample component and aparticle to the microchannel of the substrate, wherein the particlecomprises an opaque core and a shell surrounding the core.

In certain embodiments, the shell comprises a transparent material, suchas a gel. In certain embodiments, the sample component comprises a cell.In certain embodiments, the sample component and particle aresequentially added to the microchannel of the substrate. In someembodiments, the sample component and particle are added simultaneouslyto the microchannel of the substrate. In certain embodiments, the opaquecore comprises a magnetic bead.

In some aspects, the disclosure provides a method of loading a mixtureinto a microchannel of substrate comprising a plurality ofmicrochannels, the method comprising first adding to a microchannel asample component and a plurality of first particles that do not absorb awavelength X₁, and second, adding to the microchannel a plurality ofsecond particles which absorb a wavelength X₁ to the microchannel. Incertain embodiments, the plurality of second opaque particles comprisemagnetic beads.

In some aspects, the disclosure provides a method of loading a samplemixture into a microchannel of substrate comprising a plurality ofmicrochannels, the method comprising adding to a microchannel a samplematerial mixture comprising a target particle, a plurality of magneticparticles and a plurality of non-magnetic particles, and applying amagnetic force above or below the plurality of microchannels to attractthe magnetic particles to form a layer above or below the non-magneticparticles.

In certain embodiments, after applying the magnetic force, 50% or moreof the magnetic particles are not in contact with the target particle.In certain embodiments, after applying the magnetic force, 50% or moreof the magnetic particles are separated from the sample component by atleast about 1 μm or more.

In certain embodiments, the non-magnetic particles are selected fromparticles comprising silica, agarose, polystyrene or a combinationthereof. In certain embodiments, the target particles comprise intact orlysed cells. In certain embodiments, the weight ratio of magneticparticles to the non-magnetic particles in the sample material mixtureis from about 1:0.5 to about 1:10. In certain embodiments, theconcentration of magnetic particles in the sample material mixture isabout 1 mg/mL to about 30 mg/mL. In certain embodiments, theconcentration of non-magnetic particles in the sample material mixtureis about 1 mg/mL to about 100 mg/mL.

In some aspects, the disclosure provides a method of quantifying thenumber of target particles in a microchannel of a substrate, the methodcomprising first adding a sample mixture into a microchannel of asubstrate, subsequent to adding the sample mixture, adding particleslabeled with a fluorescent material to the microchannels of thesubstrate, and quantifying the number of target particles in themicrochannels.

In certain embodiments, quantifying the number of target particles inthe microchannels is performed using microscopy. In certain embodiments,the particles are opaque beads. The opaque beads may be selected fromDynabead, Agarose, and ProMag. In certain embodiments, the particles areadded about 5 minutes or more after the sample mixture is added to themicrochannels. In certain embodiments, the target particles comprise acell.

In some aspects, the disclosure provides a method of quantifying thenumber of target particles in a microchannel of a substrate, the methodcomprising adding a sample mixture comprising target particles into amicrochannel of a substrate, adding a fluorescent material to themicrochannels of the substrate, and quantifying the number of targetparticles in the microchannels by detecting the fluorescence emittedfrom the microchannels, wherein the intensity of fluorescence emitted iscorrelated to target particle count.

In certain embodiments, the fluorescent material is in solution. Incertain embodiments, the sample mixture and fluorescent material areadded concurrently to the microchannels. The fluorescent material may beassociated with the target particles. In certain embodiments, highemitted fluorescence is correlated with low target particle count or notarget particle count and low emitted fluorescence is correlated withhigh target particle count. In certain embodiments, the target particleis a cell.

In some aspects, the disclosure provides a cassette for detecting andsorting target particles, the cassette comprising a substrate with afirst surface and a second surface, wherein the second surface isconfigured to adhere a sample material mixture to the second surface, afirst housing configured to receive the substrate wherein the firsthousing comprises an internal surface to receive a target particlereleased from the substrate, a second housing coupled to the firsthousing in a manner wherein the first and second housing togetherencapsulate the substrate and wherein the first or second housingfurther comprises a first fill port, and a transmissive portion locatedin one or each of the first housing and the second housing, wherein thetransmissive portion permits transmission of electromagnetic radiationfrom outside of the cassette to the substrate.

In certain embodiments, the second surface of the substrate is at leastpartially coated to increase sample material mixture adhesion to thesecond surface. In certain embodiments, the second surface of thesubstrate is at least partially coated to increase target particleadhesion to the second surface. In certain embodiments, the substratecomprises glass.

In some aspects, the disclosure provides an apparatus to sort targetparticles, the apparatus comprising an excitation light source to emitan excitation beam to generate fluorescence light from target particleslocated on a surface or in a plurality of channels, a detector toreceive fluorescence light from the target particles, an extractionlaser to provide an extraction beam to remove target particles from thesurface or a plurality of channels, a scanner coupled to the extractionbeam to scan the excitation beam and the extraction beam to the surfaceor plurality of channels, and circuitry coupled to the detector and theextraction beam to selectively remove target particles in response tofluorescence detected from the surface or channels, wherein theapparatus is configured to process the surface or plurality of channelsat a rate within a range from about 5,000 to about 100,000,000 targetparticles per second.

In certain embodiments, the apparatus comprising an excitation lightsource to emit an excitation beam to generate fluorescence light fromtarget particles located on a surface, a detector to receivefluorescence light from the target particles, an extraction laser toprovide an extraction beam to remove target particles from the surface,a scanner coupled to the extraction beam to scan the excitation beam andthe extraction beam to the surface, and circuitry coupled to thedetector and the extraction beam to selectively remove target particlesin response to fluorescence detected from the surface, wherein theapparatus is configured to process the surface at a rate within a rangefrom about 5,000 to about 100,000,000 target particles per second.

In certain embodiments, the apparatus comprising an excitation lightsource to emit an excitation beam to generate fluorescence light fromtarget particles located in a plurality of channels, a detector toreceive fluorescence light from the target particles, an extractionlaser to provide an extraction beam to remove target particles from aplurality of channels, a scanner coupled to the extraction beam to scanthe excitation beam and the extraction beam to plurality of channels,and circuitry coupled to the detector and the extraction beam toselectively remove target particles in response to fluorescence detectedfrom the channels, wherein the apparatus is configured to process theplurality of channels at a rate within a range from about 5,000 to about100,000,000 target particles per second.

In certain embodiments, the circuitry, the extraction laser and thedetector are configured to process the surface or plurality of channelsat a rate within the range from about 25,000 to about 20,000,000 targetparticles per second. The scanner may be optically coupled to theexcitation beam and the extraction beam to scan the excitation beam andthe extraction beam together along the surface or plurality of channels.

In certain embodiments, the scanner, the excitation beam, and theextraction beam are arranged with optics to scan the excitation beam andthe extraction beam to the surface or plurality of channels with theexcitation beam separated from the extraction beam. In some embodiments,the scanner, and a plurality of extraction beams are arranged withoptics to scan the extraction beams to the surface or plurality ofchannels with the extraction beams separated from each other andindependently modulated. In certain embodiments, the optics areconfigured to simultaneously focus the excitation beam to a firstlocation on the surface or a first channel of the plurality of channelsand the extraction beam to a second location on the surface or a secondchannel of the plurality of channels, wherein the first location isseparated from the second location by a distance within a range fromabout 100 μm to about 5 mm and optionally wherein the distance is withina range from about 250 μm to about 1 mm. In certain embodiments, whereinthe scanner, the optics, the excitation beam, and the extraction beamare arranged to simultaneously focus the excitation beam to a firstlocation on the surface or a first channel of the plurality of channelsand the extraction beam to a second location on the surface or a secondchannel of the plurality of channels, wherein the first location isseparated from the second location by a distance within a range fromabout 100 μm to about 1 mm.

In certain embodiments, the scanner comprises one or more substantiallyflat mirror surfaces, and wherein the excitation beam and the extractionbeam are arranged to reflect together from each of the plurality ofsubstantially flat mirror surfaces.

In certain embodiments, the scanner comprises a first scanner to reflectand scan the excitation beam and a second scanner to reflect and scanthe extraction beam, wherein the circuitry is configured to coordinatescanning of the excitation beam with the first scanner and scanning ofthe extraction beam with the second scanner along the array toselectively remove target particles from the surface or plurality ofchannels.

In certain embodiments, the first scanner and the second scanner arelocated on one side of a substrate defining the surface or the pluralityof channels. In some embodiments, the first scanner and the secondscanner are located on opposite sides of a support defining the surfaceor the plurality of channels. In certain embodiments, the first scannerand the second scanner are independently selected from the groupconsisting of a polygonal scanner, a galvanometer scanner, an acoustooptic modulator, digital light processing system (DLPS) and a resonantscanner. In certain embodiments, the scanner is selected from the groupconsisting of a polygonal scanner, a galvanometer scanner and an acoustooptic modulator.

In certain embodiments, the circuitry and the detector are configured todetect fluorescence of a target particle above a threshold amount ineach location of the surface or each channel of the plurality ofchannels and, based on the fluorescence response, to selectivelyirradiate each location of the surface or each channel the plurality ofchannels, wherein the length of time elapsing between the fluorescenceand the irradiation lies within a range from about 10 ns to about 100μs, optionally within a range from about 100 ns to about 10 μs. Incertain embodiments, the excitation light source, the extraction laser,and the circuitry are synchronized to a shared clock. In certainembodiments, the excitation light source is configured to emit aplurality of wavelengths, each of the plurality of wavelengthscomprising a peak separated from other peaks of the plurality ofwavelengths. The excitation light source may be selected from the groupconsisting of LEDs and lasers. In certain embodiments, the opticscomprise an objective lens and wherein the excitation source, theobjective lens, and the detector are arranged in a confocalconfiguration.

In certain embodiments, the objective lens comprises an F-theta optic.In certain embodiments, the scanner comprises a mirror and the opticsare arranged to focus the excitation beam to a location of the surfacethrough the objective lens with the mirror at a first angle and totransmit first fluorescent light from a first target particle in thelocation of the surface to the detector through the objective lens withthe mirror at the first angle and wherein the optics are arranged tofocus the excitation beam to a second location of the surface throughthe objective lens with the mirror at a second angle and to transmitfluorescent light from a second target particle at the second locationof the surface to the detector through the objective lens with themirror at the second angle, in order to scan the excitation beam to aplurality of locations on the surface and measure fluorescence from theplurality of locations on the surface with the confocal configuration.

In certain embodiments, the scanner comprises a mirror and wherein theoptics are arranged to focus the excitation beam to a first channel ofthe plurality of channels through the objective lens with the mirror ata first angle and to transmit first fluorescent light from a firsttarget particle in the first channel of the plurality of channels to thedetector through the objective lens with the mirror at the first angleand wherein the optics are arranged to focus the excitation beam to asecond channel of the plurality of channels through the objective lenswith the mirror at a second angle and to transmit fluorescent light froma second target particle in the second channel of the plurality ofchannels to the detector through the objective lens with the mirror atthe second angle, in order to scan the excitation beam to a plurality ofchannels and measure fluorescence from the plurality of channels withthe confocal configuration.

In certain embodiments, the optics are arranged to transmit theexcitation beam coaxially with a field of view of the detector throughthe objective lens. In certain embodiments, a field of view of thedetector is no more than about 100 mm along a surface of a substratedefining the surface or the plurality of channels when the excitationbeam is focused on a location of the surface or a channel of theplurality of channels and optionally wherein the beam is configured toutilize diffuse infinite conjugate excitation light. In certainembodiments, the detector comprises a field of view at the surface orthe plurality of channels, and wherein a full width half maximumcross-sectional size of the beam at the surface or the plurality ofchannels is no more than the field of view at the surface or theplurality of channels and optionally wherein the full width half maximumcross-sectional size is no more than about half of the field of view. Incertain embodiments, the field of view of the detector is defined withan optical structure selected from the group consisting of an aperture,a dimension across the aperture, a pinhole, mirror, and a maximumdimension across a reflective surface across a mirror.

In certain embodiments, the detector comprises a plurality of detectorsand wherein a field of view of each of the plurality of detectors isarranged in the confocal configuration with the excitation beam. Incertain embodiments, the excitation beam comprises a plurality ofoverlapping excitation beams and wherein each of the plurality ofexcitation beams is arranged in the confocal configuration with thedetector. In certain embodiments, the excitation beam comprises a firstexcitation beam and a second excitation beam and wherein a first fieldof view of a first detector is confocal with the first excitation beamand a second field of view of a second detector is confocal with thesecond excitation beam.

In certain embodiments, a maximum cross-sectional dimension of each ofthe plurality of channels is within a range from about 10 μm to about100 μm in order to contain a single target particle. In certainembodiments, the circuitry is configured to pulse the extraction beam inresponse to fluorescence of a target particle with an amount of energysufficient to extract the target particle from the surface or thechannel and allow the target particle to survive and optionally whereinan amount of energy to extract the target particle within a range fromabout 0.1 μJ to about 1000 μJ. In certain embodiments, the circuitry isconfigured to generate a plurality of pulses to extract a plurality oftarget particles, and wherein an amount of extraction energy to eachextracted target particle is within a range from about 1 μJ to about 50μJ and wherein a duration of the extraction energy to the each extractedtarget particle is within a range from about 0.1 ns to about 1000 ns andwherein a peak extraction power to the each of the plurality ofextracted target particles is within a range from about 0.1 W to about10⁷ W.

In some aspects, the disclosure provides an apparatus to sort targetparticles, the apparatus comprising an objective lens to direct lightonto a plurality of channels sized to contain the target particles, alight source to generate an excitation beam, the light source opticallycoupled to the objective lens to generate fluorescence from the targetparticles contained in the plurality of channels, a two dimensionalarray detector optically coupled to the objective lens to receivefluorescence light from the target particles, a laser to generate anextraction beam to remove target particles from the plurality ofchannels, and a scanner optically coupled to the extraction beam to scanthe extraction beam to the plurality of channels.

In certain embodiments, the objective lens defines a first optical pathon a first side of the lens toward the surface or the plurality ofchannels and a second optical path on a second side of the objectivelens away from the surface or the plurality of channels and wherein theextraction beam, the excitation beam, and the two dimensional arraydetector are optically coupled to the objective lens along at least aportion of the second optical path. In certain embodiments, theapparatus further comprising a first beam splitter to couple theextraction beam to the objective lens and a second beam splitter tocouple the excitation beam to the objective lens and the two dimensionalarray detector to the objective lens. In certain embodiments, the firstbeam splitter comprises a dichroic coating to reflect the extractionbeam toward the objective lens, the dichroic coating located on asurface of the first beam splitter oriented toward the objective lensand wherein the second beam splitter comprises a dichroic beam splitterconfigured to reflect the excitation beam toward the objective lens andtransmit the fluorescence from the target particles passing through theobjective lens to the two dimensional array detector.

In certain embodiments, the apparatus further comprising an F-thetarelay lens to couple the extraction beam from the scanner to theobjective lens. In certain embodiments, the apparatus further comprisingan F-theta relay lens pair to couple the extraction beam from thescanner to the objective lens.

In certain embodiments, the apparatus further comprises a wavelengthselector coupled to the excitation beam to filter the excitation beambetween a beam splitter and the excitation light source and optionallywherein the wavelength selector is selected from the group consisting offilter wheel comprising a plurality of filters, a prism and a grating.

In some aspects, the disclosure provides a method of detecting andsorting target particles, the method comprising providing a substratewith a plurality of microchannels, wherein the microchannels comprise asample material mixture comprising a plurality of target particles,scanning the contents of the microchannels with an excitation beam,detecting a fluorescence signal emitted from the microchannels whereinthe fluorescence signal indicates the presence of the target particle ina microchannel, and extracting the target particles from microchannelswith an extraction beam, wherein said length of time elapsing betweensaid detecting fluorescence and extracting said target particles from asingle microchannel is from about 10 ns to about 100 μs.

In some aspects, the disclosure provides a method of detecting andsorting target particles, the method comprising providing a substratewith a plurality of microchannels, wherein the microchannels comprise asample material mixture comprising a plurality of target particles,scanning the contents of the microchannels with an excitation beam,detecting a fluorescence signal emitted from the microchannels whereinthe fluorescence signal indicates the presence of the target particle ina microchannel, and extracting the target particles from microchannelswith an extraction beam, wherein scanning said microchannels with anexcitation beam occurs at a rate greater than 1,000,000 microchannelsper second, greater than 2,000,000 microchannels per second, or greaterthan 3,000,000 microchannels per second.

In some aspects, the disclosure provides a method of detecting andsorting target particles, the method comprising providing a substratewith a plurality of microchannels, wherein the microchannels comprise asample material mixture comprising a plurality of target particles,scanning the contents of the microchannels with an excitation beam,detecting a fluorescence signal emitted from the microchannels whereinthe fluorescence signal indicates the presence of the target particle ina microchannel, and extracting the target particles from microchannelswith an extraction beam, wherein extracting the target particles fromsaid microchannels with an extraction beam occurs at a rate greater than500,000 microchannels per second, 600,000 microchannels per second,700,000 microchannels per second, 800,000 microchannels per second,900,000 microchannels per second, or 1,000,000 microchannels per second.

In some aspects, the disclosure provides a method of detecting andsorting target particles, the method comprising providing a substratewith a plurality of microchannels, wherein the microchannels comprise asample material mixture comprising a plurality of target particles,scanning the contents of the microchannels with an excitation beam,detecting a fluorescence signal emitted from the microchannels whereinthe fluorescence signal indicates the presence of the target particle ina microchannel, and extracting the target particles from microchannelswith an extraction beam, wherein extracting the target particles fromsaid microchannels results in a collection of particles with a puritygreater than 90%, greater than 95%, or greater than 99%.

In some aspects, the disclosure provides a fluorescence microscopecomprising: a fluorescence excitation light source configured to directfluorescence excitation light to a sample and to induce the emission ofemitted fluorescence light from the sample; an objective lens configuredto receive emitted fluorescence light from the sample; and a lightdetector configured to detect emitted fluorescence light received by theobjective lens. The fluorescence excitation light source may beconfigured to direct the fluorescence excitation light to the samplesuch that the fluorescence excitation light does not pass through theobjective lens. Directing the fluorescence excitation light to thesample such that the fluorescence excitation light does not pass throughthe objective lens may reduce background fluorescence detected by thelight detector. Directing the fluorescence excitation light to thesample such that the fluorescence excitation light does not pass throughthe objective lens may reduce speckle noise detected by the lightdetector.

The disclosure provides a method of sorting cells, comprising screeningcellular material to identify cells with a desired phenotype, whereinthe cellular material is screened at a rate of 100,000 cells per secondor greater. In certain embodiments, the cellular material is screened ata rate 500,000 cells per second or greater, such as at a rate of1,000,000 cells per second or greater. In certain embodiments, themethod further comprises extracting said cells of a desired phenotypefrom said cellular material at a rate of 100,000 cells per second orgreater, such as at a rate of 300,000 cells per second or greater. Saidcellular material may be screened in an array with through-holes andsaid cells of a desired phenotype may be extracted from said array. Incertain embodiments, the extracting comprises releasing said cells of adesired phenotype using electromagnetic radiation. In certainembodiments, each through-hole of said array comprises from 0 to about 5cells and at least 30% of the through-holes of the array comprise atleast one cell.

In certain embodiments, the cellular material of said method is obtainedfrom a human subject and/or comprises less than 5% of HSCs and HSPCs. Incertain embodiments, greater than 95% of the extracted cells are HSCsand/or HSPCs. In certain embodiments, greater that 95% of the extractedcells are cells of said desired phenotype. In certain embodiments,greater than 95% of the extracted cells are viable as determined byevaluating the cells within 5 hours from said extracting. In certainembodiments, the extracted cells are suitable for therapeutic usewithout the need for additional sterilization steps. The extracted cellsmay be essentially free of pathogens. The extracted cells may compriseless than 0.1% of pathogens. In certain embodiments, the disclosureprovides extracted cells obtained by any of the methods herein andpharmaceutical composition thereof.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

Features of the invention are set forth with particularity in theappended claims. A better understanding of the features and advantagesof the present invention will be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of the invention are utilized, and the accompanyingdrawings of which:

FIG. 1 is a cassette for sorting target particles according to anillustrative embodiment of the invention.

FIG. 2 represents an external view of a cassette for sorting targetparticles.

FIG. 3 represents several external and internal components of a cassettefor sorting target particles.

FIG. 4A illustrates one embodiment of adding or loading a samplematerial mixture to a sample well.

FIG. 4B illustrates one embodiment of adding or loading a samplematerial mixture from a sample well to a substrate comprising aplurality of microchannels. The sample well moves parallel to thesubstrate to add or load sample material mixture to the substrate. Thesample well may move from the first end of the substrate the second endof the substrate to add or load sample material mixture to thesubstrate.

FIG. 4C illustrates one embodiment of adding or loading a solution tothe hydration membrane of the cassette through the hydrate port on thetop cover of the cassette.

FIG. 4D illustrates one embodiment of placing a frame containing ahydration membrane on the surface of a substrate through an externalmagnetic force.

FIG. 4E illustrates one embodiment of scanning target particles throughthe bottom window of the bottom cover of the cassette.

FIG. 4F illustrates one embodiment of flushing the extracted targetparticles from the internal surface of the bottom cover to thecollection well.

FIG. 4G illustrates one embodiment of flushing and recovering theextracted target particles from the release port.

FIG. 5A represents several internal components of a cassette thatinclude a substrate, a seal, and a metal frame.

FIG. 5B represents the substrate affixed to the metal frame through aseal, which reduces sagging of the substrate under heated conditions.

FIG. 6 illustrates one embodiment of loading a sample material mixtureinto a microchannel of a substrate. The microchannel comprises a firstmixture and a second mixture. The first mixture comprises a transparentsolution and a plurality of opaque particles. The second mixturecomprises of a cellular component and an aqueous solution.

FIG. 7A represents a microchannel of a substrate that is loaded with acellular component and a plurality of opaque particles.

FIG. 7B represents a microchannel of a substrate that is loaded with acellular component and a plurality of particles comprising an opaquecore and a shell surrounding the core.

FIG. 7C is a graphical representation of extraction efficiency of cellsthat were independently loaded with either opaque particles or particlescomprising an opaque core and a shell surrounding the core. Thegraphical representation illustrates that the cells containing opaqueparticles were extracted with less efficiency than the cells containingparticles comprising an opaque core and a shell surrounding the core.

FIG. 7D is a graphical representation of cell survival of cells thatwere independently loaded with either opaque particles or particlescomprising an opaque core and a shell surrounding the core. Thegraphical representation illustrates that the cells containing opaqueparticles were less viable than the cells containing particlescomprising an opaque core and a shell surrounding the core.

FIG. 8A represents a microchannel of a substrate that is loaded with amixture comprising a cellular component, a plurality of magneticparticles, and a plurality of non-magnetic particles.

FIG. 8B is a graphical representation of extraction efficiency and rateof survival of cells that were loaded with varying amounts ofnon-magnetic particles, and a set amount of magnetic particles.

FIG. 9A represents an illustrative embodiment of the sequential loadingmethod.

FIG. 9B is a graphical representation of the number of cells permicrochannel of the substrate. The cells were loaded onto the substratecomprising a plurality of microchannels with Dynabeads® in a mixedbatch.

FIG. 9C is a graphical representation of the number of cells permicrochannel of the substrate. The cells were loaded onto the substratecomprising a plurality of microchannels with Dynabeads® in a sequentialmanner.

FIG. 9D is a graphical representation of the number of cells permicrochannel of the substrate. The cells were loaded onto the substratecomprising a plurality of microchannels with agarose in a mixed batch.

FIG. 9E is a graphical representation of the number of cells permicrochannel of the substrate. The cells were loaded onto the substratecomprising a plurality of microchannels with agarose in a sequentialmanner.

FIG. 9F is a graphical representation of the number of cells permicrochannel of the substrate. The cells were loaded onto the substratecomprising a plurality of microchannels with ProMag® in a mixed batch.

FIG. 9G is a graphical representation of the number of cells permicrochannel of the substrate. The cells were loaded onto the substratecomprising a plurality of microchannels with ProMag® in a sequentialmanner.

FIG. 9H is a graphical representation of the number of cells permicrochannel of the substrate. The cells were loaded onto the substratecomprising a plurality of microchannels with no opaque particles.

FIG. 10A illustrates the microchannels of a substrate loaded withfluorescent material and cells. The fluorescent material is displaced inthe presence of cells. The number of cells in the microchannel isquantified by the amount of fluorescent displacement. The fluorescencesignal is more intense in channels with a low cell count or is lessintense in channels with a high cell count.

FIG. 10B shows the different fluorescent intensity levels that aredetected or visualized by the presence or absence of cells. Themicrochannels of the substrate contain fluorescent material in thefluorescein isothiocyanate (FITC) channel. The relatively darkenedregions indicate the presence of one or more cells in the microchannelof the substrate. The relatively bright regions indicate the absence ofcells in the microchannel of the substrate.

FIG. 10C shows the different fluorescent intensity levels that aredetected or visualized by the presence or absence of cells. Themicrochannels of the substrate contain fluorescent material in thefluorescein isothiocyanate (FITC) channel. The relatively darkenedregions indicate the presence of one or more cells in the microchannelof the substrate. The relatively bright regions indicate the absence ofcells in the microchannel of the substrate. The regions containing cellsare circled.

FIG. 10D identifies the regions that contain cells stained with celltracker far red dye in the microchannel of the substrate. Themicrochannels containing the dye fluorescence correspond to relativelybright regions.

FIG. 10E identifies the regions that contain cells stained with the cellfar red in the microchannel of the substrate. The microchannelscontaining cell tracker far red correspond to relatively bright regions.The remaining regions correspond to microchannels containing fluorescentmaterial, but no cells.

FIG. 10F is a graphical representation of fluorescent intensity in thepresence of cells and absence of cells. The average fluorescentintensity was lower in the presence of a cell than in its absence.

FIG. 11 is a schematic of an optical apparatus for laser scanning cellsorting utilizing a rotating polygon mirror.

FIG. 12 is a schematic of an optical apparatus for laser scanning cellsorting utilizing two rotating polygon mirrors.

FIG. 13 is a schematic of an optical apparatus for laser scanning cellsorting utilizing two rotating polygon mirrors and a confocal detectiontechnique.

FIG. 14 is a schematic of an optical apparatus for laser scanning cellsorting utilizing a galvanometer scanning mechanism.

FIG. 15 shows the optimal pulse power settings for laser extraction ofcells from a microchannel array.

FIG. 16 shows an exemplary digital processing device programmed orotherwise configured to operate a laser scanning cell sorting device.

FIG. 17 is a schematic for an alternative fluorescence detection system.

DETAILED DESCRIPTION OF THE INVENTION

In a brief overview, the embodiments of the present disclosure providemethods and apparatuses for sorting target particles. In variousembodiments, the present disclosure provides methods and apparatusesthat screen, extract, and sort target particles in a simple, rapid,efficient and cost-effective manner. Additionally, in variousembodiments, the present disclosure provides methods and apparatusesthat screen, extract, and sort target particles under sterileconditions. In an exemplary embodiment, the present disclosure providesmethods and apparatuses for sorting viable cellular material.

The present disclosure envisions target particles to include a widerange of particles, such organic and inorganic particles, natural andsynthetic particles, and combinations thereof. The target particlespresented herein are exemplary embodiments of target particles and arenot limited to the particles described herein. In various embodiments,target particles may include any particle that weighs about 20 daltonsto about 200 kilodaltons. In some embodiments, target particles may beidentifiable through fluorescence. In some embodiments, target particlesmay include inorganic particles, such as metal beads and silica beads.For instance, metal beads may include beads containing alumina (e.g.,gamma alumina). In some embodiments, target particles may include silicabeads. Target particles may include polymers, such as polystyrene,polyethylene, poly(vinylpyrrolidone), acrylamidopropyl-PEG andderivatives thereof. In certain embodiments, the target particlescomprise a Merrifield resin, hydroxymethyl resin, Wang resin,aminomethyl resin, SASRIN resin, TentaGel S AC resin, TentaGel PHBresin, TentaGel S NH₂ resin or combinations thereof. Target particlesmay also comprise carbon nanotubes and fullerenes. In certainembodiments, target particles comprise particles that are used insplit-pool synthesis. In some embodiments, target particles comprisecellular material, such as whole cells, lysed cells, cellularcomponents, extracellular matrix, biological tissue, and portionsthereof. In some embodiments, target particles comprise biomoleculessuch as proteins, peptides, antibodies, carbohydrates, lipids, nucleicacids, nucleotides, primary metabolites, secondary metabolites, andnatural products. Target particles may also include small molecules,both synthetic and natural small molecules, for example, molecules whichweigh less than 1000 daltons. In some embodiments, target particles mayinclude viruses.

In certain embodiments, the target particles comprise cellular material,such as whole cells or lysed cells. Cells may be any cell derived froman organism, which include human, animal, fungal, microbial, insect, andmodified cells thereof. In certain embodiments, the target particles areidentified in a cellular material mixture. In certain embodiments, acellular material mixture comprises cellular material, an aqueoussolution and optionally opaque particles. Examples of an aqueoussolution include media, buffer, and water. In certain embodiments, thedisclosure provides systems and methods to isolate target cells from acellular material mixture, wherein said target cells express or produceparticular proteins, carbohydrates, enzymes, peptides, hormones,receptors, or combinations thereof. In certain embodiments, thedisclosure provides systems and methods to isolate target cells from acellular material mixture that that produce particular antibodies. Incertain embodiments, the disclosure provides systems and methods toisolate target cells from a cellular mixture that are particulargenetically engineered cells or activated cells.

The term “opaque,” as described herein, refers to a material thatabsorbs at least a portion of the electromagnetic spectrum. An opaquematerial may not permit, at least partially, the passage of visiblelight through the material. An opaque material may not permit thepassage of one or more wavelengths ranging from about 390 nm to about700 nm. For example, such materials will not permit the passage of oneor more wavelengths ranging from about 450 nm to about 495 nm.

In one aspect, the present disclosure is directed to a cassette forsorting target particles. In some embodiments, the cassette is anenclosed system that permits the sorting of target particles understerilized conditions. The cassette may be sterilized prior to use. Thecassette may contain one or more fill ports that allow the user tointroduce a sample material mixture and optionally other solutions intothe cassette without compromising sample integrity, e.g., exposing thesample to pyrogens or other contaminants. In some embodiments, thecassette may be intended for only a single use, e.g., disposable afteruse. For example, the cassette may be used for sorting cellular materialfrom a single patient sample, and disposed of after use. Additionally,the cassette may be configured to be received by various apparatuses andmachines to facilitate sorting of target particles. In anotherembodiment, the cassette may be available with instructional informationon how to use the cassette.

In another aspect, the disclosure provides a method for sorting targetparticles using the cassette of the disclosure. In various embodiments,a sample material mixture may be loaded into the microchannels of asubstrate of a cassette for scanning and extraction of target particles.In an exemplary embodiment, a cellular material mixture may be loadedinto the microchannels of a substrate of a cassette for scanning andextraction of cellular material. In certain embodiments, the samplematerial mixture may be loaded onto the first surface of the substratefor scanning and extraction. In certain embodiments, one or more typesof cells, e.g., cells with certain cell surface markers, may beextracted from a cellular material mixture. In certain embodiments, oneor more types of cells that are secreting certain proteins or otherbiomolecules of interest may be extracted from a cellular materialmixture. In certain embodiments, one or more types of cells that areexpressing certain intracellular proteins or other biomolecules ofinterest may be extracted from a cellular material mixture. In certainembodiments, one or more types of cells that are expressing certainproteins or other biomolecules of interest targeted to a specificorganelle or other subcellular localization may be extracted from acellular material mixture. In certain embodiments, one or more types ofcells that are expressing a combination of the aforementioned attributesmay be extracted from a cellular material mixture. In certainembodiments, the extraction of one or more types of cells may beperformed under sterile conditions.

In certain embodiments, the microchannels of the substrate may compriseopaque particles. The opaque particles may be involved in a process inwhich the opaque particles absorb electromagnetic radiation and,vaporize a portion of the aqueous media, which thereby causes thecellular material in the microchannel to be released from themicrochannel. In certain embodiments, the energy released from saidprocess may damage the cellular viability of the cellular material. Toprotect the viability of the cellular material, the opaque particles maybe added to the microchannels in a manner that protects and preservescellular viability of the cellular material from said process. Incertain embodiments, the viability of the cellular material ismaintained by spacing the opaque particles a distance away from cells inthe microchannels. In one embodiment, the method involves the additionof two mixture layers to the microchannel of substrate, wherein thefirst mixture layer contains opaque particles in a transparent solution,e.g., suspended in a gel, and the second mixture layer contains cellularmaterial in an aqueous solution. In certain embodiments, the method ofloading microchannels of a substrate involves the addition of particlescomprising an opaque core and an insulating outer shell of a transparentmaterial, which prevents contact or close approach of a cell with theopaque core. In certain embodiments, the method of loading microchannelsof a substrate involves the addition of opaque microparticles andtransparent microparticles, in which the transparent microparticlesprevent contact or close approach of the cell with the opaquemicroparticles.

Additionally, the disclosure provides a method to position cells closerto the opening of the microchannel than the opaque particles. In someembodiments, cells are observed through one opening of the microchannel,and it is advantageous over existing methods to position the opaqueparticles distal to the cell from the plane of observation. Suchpositioning of the opaque particles may permit the observation of agreater number of cells relative to the number of cells loaded onto themicrochannel. In certain embodiments, the method of loadingmicrochannels of a substrate involves the addition of a cellularmaterial mixture and opaque particles in a sequential manner.

Additionally, the disclosure provides a method to control the positionthe cells and opaque material within the microchannel. In someembodiments, cells are assisted towards one end of the microchannelusing sonication or ultra-sonication. In some embodiments the opaquematerial is assisted towards one end of the microchannel usingsonication or ultra-sonication. In some embodiments, these processes areperformed sequentially. In certain embodiments, a cassette of thedisclosure further comprises a contact transducer or similar device,wherein the contact transducer may be located at any functional positionon the cassette, e.g., in contact with the substrate within the housingor on the external surface of the housing. In certain embodiments, thecontact transducer may be in contact with one or more components of acassette described herein.

Additionally, the disclosure provides a method to discriminate one cellfrom multiple cells deposited in microchannels of a substrate. Incertain embodiments, the method involves the use of fluorescent materialpositioned distal to the cell or cells from the plane of observation.The fluorescent light of the fluorescent material may be subsequentlymeasured. In certain embodiments, the method involves the distributionof fluorescent material amongst the cellular material and thedisplacement of fluorescent material in the presence or absence of cellsis used to quantify cellular material.

Additionally, the disclosure provides optical apparatuses for laserscanning cell sorting.

In one embodiment, the disclosure provides an optical apparatusutilizing a rotating polygon mirror. In a further embodiment, saidapparatus is combined with a linear stage, X-Y stage, or galvanometers.

In another embodiment, the disclosure provides an optical apparatusutilizing two rotating polygon mirrors. In a further embodiment, saidapparatus is combined with one or more linear stages, X-Y stages,galvanometers, digital light processing systems, or resonant scanners.

In another embodiment, the disclosure provides an optical apparatusutilizing two rotating polygon mirrors and a confocal detectiontechnique. In a further embodiment, said apparatus is combined with alinear stage, X-Y stage, or galvanometers.

In another embodiment, the disclosure provides an optical apparatusutilizing a galvanometer scanning mechanism. In a further embodiment,said apparatus is combined with a linear stage, X-Y stage, orgalvanometers.

In some aspects, the methods of the disclosure enable the preparation ofpharmaceutical compositions of hematopoietic stem cells (HSCs) and/orhematopoietic stem progenitor cells (HSPCs) with unprecedentedsterility, purity, and viability. In particular, the disclosure providesmethods of sorting cells, e.g., cells obtained from a subject, whereinthe method comprises one or more of the following: (a) screeningcellular material at a rate of 100,000 cells per second or more, such as200,000 cells per second or more, such as 300,000 cells per second ormore; (b) scanning cells wherein less than 10% of the original cellularmaterial comprises HSCs and/or HSPCs (c) extracting cells of a desiredphenotype or phenotypes at a rate of 100,000 cells per second or more,such as 200,000 cells per second or more, such as 300,000 cells persecond or more; (d) extracting cells thereby producing a cell extractwherein 95% or more of said cell extract cells are HSCs and/or HSPCs;(e) extracting cells thereby producing a cell extract wherein 95% ormore, such as 95% or more or even 98% or more of the extracted HSCsand/or HSPCs have the desired phenotype or phenotypes; (f) extractingcells thereby producing a cell extract wherein the cell extract has aviability of greater than 95%; and (g) extracting cells therebyproducing a cell extract wherein HSCs and/or HSPCs of the cell extractare suitable for clinical use without the need for further purificationor sterilization.

In some aspects, the disclosure provides pharmaceutical compositions ofHSCs and/or HSPCs with one or more of the following characteristics: (a)the composition comprises less than approximately 1% of pathogens andother contaminants, such as a negligible amount of pathogens and othercontaminants; (b) 95% or more, such as 95% or more or even 98% or moreof the HSCs and/or HSPCs of the composition have a desired phenotype orphenotypes; (c) 95% or more of the cells of the composition are viable;(d) the composition comprises less than 0.01% of naïve T cells; and (e)the composition is suitable for therapeutic use.

Definitions

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

Unless otherwise defined, the technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Expansion and clarification of some terms are provided herein.All publications, patent applications, patents and other referencesmentioned herein, if not otherwise indicated, are explicitlyincorporated by reference.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

The term “hydration” and “hydrate” as used herein refers to restoring ormaintaining fluid balance.

The term “sterile” and “sterilized” as used herein refers to reduce thepresence of contaminants, bacteria, pathogens or other unwanted livingorganisms.

In the figures presented herein, like numbered elements refer to likecomponents.

Cassette

In an exemplary embodiment, FIG. 1 illustrates a cassette for sortingtarget particles. The present disclosure envisions target particles toinclude a wide range of particles, such organic and inorganic particles,natural and synthetic particles, and combinations thereof. The targetparticles presented herein are exemplary embodiments of target particlesand are not limited by the exemplary embodiments. In variousembodiments, target particles may include any particle that weighs about20 daltons to about 200 kilodaltons. In some embodiments, targetparticles may be identifiable through fluorescence. In some embodiments,target particles may include inorganic particles, such as metal beadsand silica beads. For instance, metal beads may include beads containingalumina (e.g., gamma alumina). In some embodiments, target particles mayinclude silica beads. Target particles may include polymers, such aspolystyrene, polyethylene, poly(vinylpyrrolidone), acrylamidopropyl-PEGand derivatives thereof. For example, the polymers may be a Merrifieldresin, hydroxymethyl resin, Wang resin, aminomethyl resin, SASRIN resin,TentaGel S AC resin, TentaGel PHB resin or TentaGel S NH₂ resin. Targetparticles may also include carbon nanotubes and fullerenes. Such targetparticles may include particles that are used in split-pool synthesis.Additionally, in some embodiments, target particles may include cellularmaterial. Cellular material may include whole cells, lysed cells,cellular components, extracellular matrix, biological tissue, andportions thereof. In some embodiments, target particles may also includebiomolecules, which include proteins, peptides, antibodies,carbohydrates, lipids, nucleic acids, nucleotides, primary metabolites,secondary metabolites, and natural products. Target particles may alsoinclude small molecules, both synthetic and natural small molecules,which weigh less than 1000 daltons. In some embodiments, targetparticles may include viruses. Cellular material may comprise a cellsuspension. Cells may be any cell derived from an organism, whichinclude human, animal, fungal, microbial, insect, and modified cellsthereof. A cellular material mixture comprises cellular material, opaqueparticles, and an aqueous solution. Examples of an aqueous solutioninclude media, buffer, and water. The device of the present disclosuremay be used to isolate cells that differentially express or produceproteins, carbohydrates, enzymes, peptides, hormones, receptors, andadditionally cells that produce antibodies, genetically engineeredcells, and activated cells. In an exemplary embodiment, according toFIGS. 2 and 3, the cassette 100 comprises a top cover 110, a bottomcover 120, a substrate 130, a sample well 140, and a frame 150. Asdepicted in FIG. 2, the top cover 110 is positioned above the bottomcover 120 and is positioned in a substantially parallel plane with thebottom cover 120. The cassette 100 may be an enclosed system.

In one embodiment of a cassette for sorting target particles, asdepicted in FIG. 2, the top cover 110 may comprise one or more fillports for receiving solutions into the cassette 100. In someembodiments, the top cover may comprise one or more outlet ports fordraining solutions from the cassette. The one or more fill ports may beconfigured to introduce solutions into the cassette 100. Such solutionsmay be introduced in order to hydrate the membrane, receive a samplematerial mixture for loading a sample material mixture onto thesubstrate, receive a sample material mixture into the sample well,and/or receive buffer solution to drain target particles, such ascellular material, out of the cassette.

In certain embodiments, the top cover comprises one fill port configuredto receive solutions into the cassette. In an exemplary embodiment, asdepicted in FIG. 2, the fill ports of the top cover 110 comprise anaccessory material port 112, a hydrate port 113, and a sample port 114.The accessory material port 112, the hydrate port 113, and the sampleport 114 may be configured to receive solutions. The sample port 114 maybe configured to receive a sample material mixture for loading into asample well 140. The sample port 114 may be in fluid communication withthe sample well 140. In another embodiment, the sample port may beconfigured to receive sample material mixture for direct loading ontothe substrate and into the microchannels of the substrate. In someembodiments, about 1×10⁶ to 100×10⁹ cells are loaded into the cassette100. The hydrate port 113 may be configured to receive solutions forhydrating the hydration membrane 151. The hydrate port 113 may be influid communication with the hydration membrane 151. The accessorymaterial port 112 may be configured to receive an aqueous solution forcollecting target particles from the cassette 100. In some embodiments,the aqueous solution is a buffer. The accessory material port 112 may bein fluid communication with the internal surface of the bottom cover120, the collection well 122, and/or the receiving port 123.Additionally, the collection well 122 may be in fluid communication withthe receiving port 123. In certain embodiments, the method of loadingmicrochannels of any substrate described herein, may be accompanied by asonication step. The sonication step may allow the target particles,opaque particles or other components of the sample material mixture tosettle into the microchannels. In certain embodiments, the sonicationstep may occur prior to the scanning of the microchannels. For example,the sonication step may occur prior to, concurrent with, or subsequentto said loading step or a combination thereof. In certain embodiments,the sonication step occurs prior to and concurrent with the loading ofthe microchannels. In certain embodiments, the sonication step occursconcurrent with and subsequent to the loading of the microchannels.

In certain embodiments, the bottom cover may comprise one or more fillports for receiving solutions into the cassette. In some embodiments,the bottom cover may comprise one or more outlet ports for drainingsolutions from the cassette. The one or more fill ports may beconfigured to introduce solutions into the cassette. Such solutions maybe introduced in order to hydrate the membrane, receive a samplematerial mixture for loading a sample material mixture onto thesubstrate, receive a sample material mixture for loading a samplematerial mixture into the sample well, and/or receive buffer solution todrain target particles, such as cellular material, out of the cassette.In certain embodiments, the bottom cover comprises one fill portconfigured to receive solutions into the cassette. In anotherembodiment, the fill ports of the bottom cover comprise an accessorymaterial port, a hydrate port, and a sample port. The sample port may beconfigured to receive a sample material mixture for loading into asample well. The sample port may be in fluid communication with thesample well. In another embodiment, the sample port may be configured toreceive a sample material mixture for direct loading onto the substrateand into the microchannels of the substrate. In some embodiments, about1×10⁶ to 100×10⁹ cells are loaded into the cassette. The hydrate portmay be configured to receive solutions for hydrating the hydrationmembrane. The hydrate port may be in fluid communication with thehydration membrane. The accessory material port may be configured toreceive an aqueous solution for collecting target particles from thecassette. In some embodiments, the aqueous solution is a buffer. Theaccessory material port may be in fluid communication with the internalsurface of the bottom cover, the collection well, and/or the receivingport. Additionally, the collection well may be in fluid communicationwith the receiving port.

In various embodiments, the cassette may contain a transmissive portionthat is at least partially transparent to certain wavelengths ofelectromagnetic radiation. In an embodiment, the transmissive portion isat least partially transparent to wavelengths in the range of 250 nm to1600 nm. A transmissive portion contains a material that at leastpartially permits the transfer of one or more electromagnetic waves fromone location to another. For example, a transmissive portion mayinclude, but is not limited to, glass, quartz, plastics, or combinationsthereof. In various embodiments, the transmissive portion is atransmissive window. In some embodiments, as depicted in FIG. 3, the topcover may comprise a top window 111. The top window 111 may be atransmissive window. In another embodiment, the top window 111 may be anon-transmissive window. A non-transmissive window contains a materialthat does not permit the transfer of electromagnetic radiation from onelocation to another. In an exemplary embodiment of a cassette forsorting target particles, as depicted in FIG. 3, the bottom cover 120may comprise a bottom window 121. In another embodiment, the bottomwindow 121 may be a transmissive window. The bottom window 121 may be anon-transmissive window. In certain embodiments, the top window 111 canbe a non-transmissive window and the bottom window can be a transmissivewindow.

In an embodiment of a cassette for sorting target particles, asrepresented by FIG. 3, the cassette 100 is configured to receive asubstrate 130 within the cassette 100. The substrate is located betweenthe top cover 110 and the bottom cover 120. As shown in FIG. 1, thesubstrate is encapsulated by the housing units of the cassette 100. Thesubstrate is protected from contamination during the sorting of thetarget particles.

In certain embodiments, the cassette comprises a first housing and asecond housing that are coupled to each other in any manner known in theart. For example, the first housing and second housing may be twoseparate components that are coupled to each other through a hinge thatpermits opening of the housing to expose the inside of the cassette. Incertain embodiments, the first and second housing are two separatecomponents that are coupled to one another in a reversible manner suchas interlocked together with complementary adjoining elements, e.g., theinterface of the first housing comprises a receiving element, such as aconcave portion, and the interface of the second housing comprises adonating element, such as a convex portion, wherein the concave andconvex elements reversibly interlock with one another. In certainembodiments, the first housing and second housing are attached to oneanother in a permanent manner such as with an adhesive or meldedtogether. In certain embodiments, the first housing and second housingare coupled in a manner that they a single housing, such as where thefirst housing and second housing are not formed from the attachment ofmultiple components or are molded or formed by the stacking of layers,e.g., 3D printing, and are irreversibly coupled, e.g., do not compriseseems or hinges to separate the first housing from the second housing.

Encapsulate or encapsulation of the substrate as described herein,refers to the positioning of the entirety of the substrate within thehousing of the cassette.

In certain embodiments, the substrate contains a first surface and asecond surface. The substrate may have dimensions of about 3 mm×3 mm×0.3mm to about 5000 mm×15000 mm×1000 mm. The distance from the firstsurface to the second surface of the substrate is from about 10 μm toabout 1000 mm. In some embodiments, the substrate may be rectangular,octagonal, or circular in shape. In some embodiments, the substrate maybe a glass plate.

In certain embodiments, the first surface of the substrate is coupled tothe first surface of an absorbent material. The first surface of thesubstrate may be modified to enhance the adhesive properties of thesubstrate. For example, the first surface of the substrate may be coatedwith one or more diaminopropyl silane groups. The absorbent material maycomprise opaque particles. The absorbent material may comprise a metal.In some embodiments, the second surface of the absorbent material may becoupled to the first surface of a transparent layer. The transparentlayer may comprise agarose, collagen, matrigel, alginate andcombinations thereof. In some embodiments, a sample material mixture maybe coupled to the second surface of the transparent layer. In someembodiments, the substrate may be coupled to a channel that isconfigured to receive extracted target particles. For example, thechannel may be a flow cell tube.

In certain embodiments, the disclosure provides a substrate such asthose previously described herein with a plurality of microchannels,wherein microchannels of the substrate comprise target particles andopaque material wherein said target particles and opaque material areseparated with a spacer comprising a transparent gel or transparentsolid. In certain embodiments, the opaque material comprises a particleor a coating. In certain embodiments, the opaque material is separatedfrom the target particles by at least about 1 μm or more.

In various embodiments, the substrate may be a glass micropore array. Incertain embodiments, the substrate further comprises a plurality ofmicrochannels that extend from the first surface and second surface. Themicrochannels of the substrates described herein may be positionedsubstantially in parallel to each other. The substrate may contain about1 million to about 300 billion microchannels. In certain embodiments,microchannels of a substrate described herein have an average internaldiameter of about 50 nm to about 500 μm. In certain embodiments,microchannels of a substrate described herein have an average internaldiameter of about 50 nm to about 10 microns. In certain embodiments,microchannels of a substrate herein have an average internal diameter ofabout 50 microns to about 500 microns.

In certain embodiments, a microchannel of the substrate described hereinis capped or covered at one of the openings. In certain embodiments,microchannels of the substrate do not have an opening on one of thesurfaces of the substrate such as on the first surface or second surfaceof the substrate. A microchannel of the substrate may be capped orcovered with a material. For example, such material may beagarose-based, glass-based, metal-based, gelatin-based, and/orplastic-based.

In certain embodiments, the microchannels of the substrate areconfigured to receive a sample material mixture from the sample port. Inanother embodiment, the microchannels of the substrate 130 areconfigured to receive a sample material mixture from the sample well.The contents in the microchannels may be held in place by hydrostaticforces.

In an embodiment of a cassette for sorting target particles, as depictedin FIG. 2, the cassette 100 is configured to receive a frame 150 withinthe cassette 100. In certain embodiments, the frame 150 may furtherinclude a hydration membrane 151 within the frame 150. The frame 150containing the hydration membrane 151 may be placed on top of thesubstrate 130 following the adding or loading of a cellular materialmixture to the substrate 130. The hydration membrane 151 may seal thetop surface of the substrate 130 in order to maintain the water contentor certain concentrations of dissolved solids in the microchannel. Oneor more substantially gas permeable and/or impermeable hydrationmembranes may be used to seal the surfaces of the substrate 130following the addition of cellular material to the substrate 130. Thehydration membrane 151 can be a solid capable of retaining moisture. Forexample, the hydration membrane 151 may be a paper towel, Kimwipes®,agarose, nitrocellulose or plastics, such as polyvinylidene fluoride(PVDF). In some embodiments, a hydration layer made out of an aqueoussolution may be used instead of or in conjunction with a hydrationmembrane. In an embodiment, the hydration membrane allows water from areservoir to equilibrate with the top liquid layer of the liquid in themicrochannel, which can help mitigate the water lost from evaporation.For example, a hydration membrane 151 placed in contact with the topsurface of the substrate 130, with water placed on top of the film,would trap the contents of the microchannel within each individualmicrochannel, but would allow water or media to flow into themicrochannel. The hydration membrane may be nitrocellulose and NAFION®membranes. A similar arrangement could be obtained with a porous form ofa polytetrafluoroethylene membrane (e.g., GORE-TEX® fabrics) having verysmall holes (e.g., 10-100 nm) that would trap any cells in themicrochannel but allow water, media and other reagents to pass into themicrochannels.

In an embodiment of a cassette for sorting target particles, depicted inFIG. 2, the cassette contains a sample well 140. The sample well 140 isconfigured to receive solutions from one or more fill ports. As depictedin FIG. 4A, in one embodiment, the sample well 140 is configured toreceive a sample material mixture from the sample port 114. The samplewell may comprise, contain, or be connected to materials that may bemagnetized. Such materials include iron, nickel, cobalt, some alloys ofrare metals, some naturally occurring minerals, and compositionsthereof. In one embodiment, as depicted in FIG. 4B, the sample well 140may be configured to be in contact with a surface of the substrate 130.In certain embodiments, the sample well 140 may be configured to moveacross a surface of a substrate 130. The sample well 140 may beconfigured to move manually, mechanically, or electronically. In someembodiments, the sample well may be connected to wheels or other ballbearing which are, in turn, in contact with a smooth track attached tothe top cover. In various embodiments, the sample well may be connectedto gears, which are in turn connected to groves that are connected tothe top cover. When force is applied in parallel to the track, thesample well is guided in the direction of the tracks. Force may beapplied by a tensile force of attached cables which are connected to amotor. The motor may be internal or external to the cassette and poweredmanually with a crank or electronically with a battery or other voltagesource. In certain embodiments, the movement of the sample well may becontrolled magnetically. The sample well may be externally controlled bymagnetic forces located outside of the cassette, e.g., within theparticle sorting apparatus of the disclosure. In certain embodiments,magnets are attached to the sample well in proximity to the inner wallof the top cover. Magnetic fields external to the cassette are thenmanipulated into close contact with the internal magnets on the samplewell. Movement of the external magnetic field generates a force on themagnets on the interior of the cassette and moves the sample well alonga track on the interior of the top cover. Magnets may be rare earthmagnets like neodymium or electromagnets. In certain embodiments, asdepicted in FIG. 4B, the sample well 140 may be moved while the topcover 110 remains attached to the bottom cover 120. In one embodiment,the sample well may be configured to add or load cellular material intothe microchannels of a substrate. In one embodiment, the sample wellfurther includes a spreading implement located at the bottom surface ofthe sample well. The spreading implement may be in contact with thesubstrate to distribute cellular material into the microchannels. Thespreading instrument may resemble a squeegee, foil, or policeman. Thespreading instrument may be made out of rubber, plastic, metal or otherliquid impermeable, bio-compatible material.

The embodiment of FIG. 1 further includes a seal 510 and a metal frame520, as depicted in FIG. 5A and FIG. 5B. As depicted in FIG. 5A and FIG.5B, in one embodiment, the seal 510 is placed between the substrate 130and the metal frame 520. The seal 510 may be an adhesive material. Forexample, the seal 510 may be an epoxy adhesive, including polyurethane,acrylic, and cyanoacrylate, which can be used as an adhesive for wood,metal, glass, stone, and plastics. The epoxy adhesive can be madeflexible or rigid, transparent or opaque, fast-setting or slow setting.As depicted in FIG. 5B, the substrate 130 and the metal frame 520 are incontact with the seal 510. In one embodiment, the substrate 130, theseal 510, and the metal frame 520 may be assembled at a temperature of15° C. or less. For example, the substrate 130, the seal 510, and themetal frame 520 may be assembled at a temperature of 5° C. Asrepresented in FIG. 5B, the substrate 130, the seal 510, and the metalframe 520 are assembled when in operation. The temperature of thesubstrate 130, the seal 510, and the metal frame 520 may be increasedduring the operation of the cassette 100. At increased temperatures, thesubstrate 130, the seal 510, and the metal frame 520 may expand. In someembodiments, at increased temperatures, the expansion of the metal frameexceeds the expansion of the substrate, thereby causing a tension to beapplied across the surface of the substrate. Such tension across thesurface of the substrate may reduce sagging of the substrate and therebymaintain planarity of the substrate. In some embodiments, the substratecomprises of a first end, a second end, and a middle portion, and thefirst end, the second end, and the middle portion are substantially onthe same plane.

In an embodiment of a cassette for sorting target particles, depicted inFIG. 2, the bottom cover 120 may also comprise an internal surface forcollecting target particles. In one embodiment, the internal surface mayinclude a capture surface. The capture surface may be removable oraffixed to the cassette. For example, the capture surface may be a dish,a plate, a curved surface, or a flat surface. In some embodiments, asdepicted in FIG. 2, the internal surface of the bottom cover 120 maycomprise a collection well 122, and a receiving port 123. The internalsurface of the bottom cover 120 may be configured to receive solutionsfrom one or more fill ports from the top cover 110. The bottom cover maycomprise one or more outlet ports for draining target particles andaqueous solutions. In an embodiment of the disclosure, as depicted inFIG. 4F the internal surface of the bottom cover 120 is configured toreceive an aqueous solution from the accessory material port 112. Theaqueous solution may assist in transporting target particles from theinternal surface of the bottom cover 120 to the collection well 122. Theaqueous solution may also assist in transporting target particles fromthe collection well 122 to the receiving port 123. In one embodiment,the target particles may be recovered from the receiving port 123.

In another embodiment, the internal surface of the bottom cover isconfigured to receive solutions from one or more fill ports from thebottom cover. The bottom cover may comprise one or more outlet ports fordraining target particles and aqueous solutions. In one embodiment, theinternal surface of the bottom cover is configured to receive an aqueoussolution from a fill port. The aqueous solution may assist intransporting target particles from the internal surface of the bottomcover to the collection well. The aqueous solution may also assist intransporting target particles from the collection well to the receivingport. In certain embodiments, target particles may be recovered from thereceiving port.

In an embodiment of a cassette for sorting target particles, depicted inFIG. 1, the top cover 110 may attach to the bottom cover 120. Asdiscussed earlier, the cassette 100 may be an enclosed system. Theenclosed system protects the cellular material from contaminants. Thecassette may be sterilized prior to adding or loading of cellularmaterial onto the substrate 130. The interior portions of the cassette100 may be sterilized separately and the cassette 100 assembled understerile conditions. The top cover 110 and the bottom cover 120 mayprevent contaminant entry into the cassette 100. The top cover 110 andthe bottom cover 120 may protect the target particles fromcontamination, which include bacteria, molds, yeasts, viruses, andmycoplasma. The closed nature of the cassette 100 also protects theoperator from any potential pathogens in the sorting material, andprotects each sample from contamination from another sample.

Method of Sorting Target Particles

In various embodiments, the present disclosure provides a method forsorting target particles. In various embodiments of a method for sortingtarget particles, as depicted in FIGS. 4A and 4B, a sample materialmixture is added or loaded into the cassette 100 through a sample port114 located on the top cover 110. In some embodiments, about 1×10⁶ to100×10⁹ cells are loaded into the cassette 100. In various embodiments,the sample material mixture is directly added or loaded onto thesubstrate. In some embodiments, the substrate may be flooded with thesample material mixture. The sample material mixture may move into themicrochannels of the substrate through capillary action. In someembodiments, the substrate may be flooded with the sample materialmixture when the diameter of the microchannels ranges from about 10 nmto about 100 μm. In certain embodiments, any substrate described hereinmay further comprise border elements, wherein said border elementsextend vertically from the perimeter of the top surface of the substrateand permit containment of fluid on the top surface of said substrate. Incertain embodiments, wherein said substrate is flooded with a volume ofsample material mixture, said border elements contain said samplematerial mixture and prevent said mixture from contaminating otherportions of said cassette. In certain embodiments, the substrate maycomprise border elements with vertical dimensions from about 10 micronsto about 10 cm. In certain embodiments, the average diameter of themicrochannels of the substrate are proportional to the height of theborder elements, such that a substrate with microchannels of narrowaverage diameters, e.g., about 50 nm to about 10 microns, has borderelements with vertical dimensions from about 100 microns to about 3 mmand a substrate with microchannels of wider average diameter, e.g.,about 50 microns to about 500 micron, has border elements with verticaldimensions from about 1 mm to about 10 cm.

In another embodiment, the microchannels of the substrate may be filledwith the sample material mixture by placing a hanging drop from the topcover to a position above the microchannel. In some embodiments, thehanging drop may be in contact with the microchannel. In someembodiments, the hanging drop may range in volume from about 10 μL toabout 900 mL. The sample material mixture may move into themicrochannels through capillary action. In another embodiment, thesample material mixture may be received by the sample well 140. In anexemplary embodiment, as depicted in FIG. 4B, the sample well 140 maymove across the top surface of the substrate 130 from one end to theopposite end of the substrate. The sample well 140 may load or addsample material mixture to the substrate 130 and the microchannels ofthe substrate. The sample well 140 may load an approximately equivalentamount of sample material mixture into each microchannel of thesubstrate. In some embodiments, the sample material mixture may beloaded with a sample well when the diameter of the microchannels rangesfrom about 100 μm to about 500 μm. In some embodiments, the sample well140 may contain a spreading implement at the bottom of the sample well140. The spreading implement, may be in contact with the substrate 130to distribute sample material mixture into the microchannels and off ofthe surface of the substrate. The spreading instrument may move acrossone end to the opposite end of the substrate 130 to distribute samplematerial mixture into the microchannels of the substrate. In oneembodiment, the method of the present disclosure contemplates adistribution of cellular material that may be 1 to 1000 cells, 1 to 500cells, 1 to 100 cells, 1 to 50 cells or about 1 to 5 cells in amicrochannel of the substrate.

In another embodiment, a sample material mixture is added or loaded intothe cassette through a fill port located on the bottom cover. In someembodiments, about 1×10⁶ to 100×10⁹ cells are loaded into the cassette.In various embodiments, the sample material mixture is directly added orloaded onto the substrate. In some embodiments, the substrate may beflooded with the sample material mixture. The sample material mixturemay move into the microchannels of the substrate through capillaryaction. In some embodiments, the substrate may be flooded with thesample material mixture when the diameter of the microchannels rangesfrom about 10 nm to about 100 μm. In another embodiment, themicrochannels of the substrate may be filled with the sample materialmixture by placing a hanging drop from the bottom cover to a positionabove the microchannel. In some embodiments, the hanging drop may be incontact with the microchannel. In some embodiments, the hanging drop mayrange in volume from about 10 μL to about 900 mL The sample materialmixture may move into the microchannels through capillary action. Inanother embodiment, the sample material mixture may be received by thesample well. The sample well may move across the top surface of thesubstrate from one end to the opposite end of the substrate. The samplewell may load or add sample material mixture to the substrate and themicrochannels of the substrate. The sample well can load anapproximately equivalent amount of sample material mixture into eachmicrochannel of the substrate. In some embodiments, the sample materialmixture may be loaded with a sample well when the diameter of themicrochannels ranges from about 100 μm to about 500 μm. In someembodiments, the sample well may contain a spreading implement at thebottom of the sample well. The spreading implement, may be in contactwith the substrate to distribute sample material into the microchannels.The spreading instrument may move across one end to the opposite end ofthe substrate to distribute sample material mixture into themicrochannels of the substrate and off of the surface of the substrate.In certain embodiments, the method of the present disclosurecontemplates a distribution of cellular material that may be 1 to 1000cells, 1 to 500 cells, 1 to 100 cells, 1 to 50 cells, and about 1 to 5cells in at least one of the microchannels of the substrate.

After the sample material mixture is loaded into the microchannels ofthe substrate 100 or loaded onto the first surface of the substrate, themicrochannels and the first surface of the substrate are scanned, asdepicted in FIG. 4E, to detect one or more target particles. Scanningincludes illuminating the microchannels with a specific wavelength(first wavelength) or a set of specific wavelengths, and detecting thetarget particles with a specific wavelength (second wavelength) or a setof specific wavelengths. For example, the specific wavelengths includewavelengths ranging from about 200 nm to about 1.5 mm. The specificwavelengths for illuminating and detecting may be the same or different.Any wavelength referred to herein, e.g., first wavelength, secondwavelength, third wavelength, fourth wavelength, and wavelength X₁, maybe independently selected from at least one wavelength within a range ofthe electromagnetic spectrum extending from the ultraviolet to the farinfrared. The first, second, third, and fourth wavelengths andwavelength X₁ are independently selected from one or more wavelengthsranging from about 200 nm to about 1.5 mm. In some embodiments, thefirst, second, third, and fourth wavelengths and wavelength X₁ areindependently selected from wavelengths ranging from about 400 nm toabout 700 nm. For example, illuminating wavelengths may include 350 nmto 400 nm to illuminate 4′, 6-diamidino-2-phenylindole (DAPI); 400 nm to450 nm to excite dyes such as BV421™, cyan fluorescent protein (CFP),AmCyan, and Pacific Blue™; 450 nm to 500 nm to excite dyes such as greenfluorescent protein (GFP), Peridinin Chlorophyll Protein Complex(PerCP), and PerCP-Cy™5.5; 500 nm to 600 nm to illuminate dyes such asR-phycoerythrin (PE), PE-Texas Red®, Texas Red®, 7-aminoactinomycin D(7-AAD), PE-Cy™5, PE-Cy™5.5, PE-Cy™7, and PE-Dazzle™; 600 nm to 700 nmto excite dyes such as allophycocyanin (APC), Alexa Fluor® 647, AlexaFluor® 700, Alexa Fluor® 780, and APC-Cy™7; and 800 nm to 1200 nm toilluminate AG₂SE Quantum dots or single-walled carbon nanotubes (SWNT).Detection wavelengths may include 350 nm to 400 nm and 400-500 nm todetect a dye such as BV421™; 500 nm to 600 nm to detect dyes such asgreen fluorescent protein (GFP) and Peridinin Chlorophyll ProteinComplex (PerCP); 600 nm to 700 nm, 800 nm to 900 nm, 900 nm to 1100 nm,and 1100 nm to 1300 nm to detect single-walled carbon nanotubes (SWNT)and AG₂SE Quantum dots; and 1300 nm to 1500 nm to detect AG₂SE Quantumdots and single-walled carbon nanotubes (SWNT). In addition, theelectromagnetic radiation source may illuminate one wavelength as in alaser source or a band of wavelength as in LED light sources. Theelectromagnetic radiation source may be used alone, or a light path maybe generated containing multiple wavelengths or multiple powerssimultaneously. Different wavelengths or powers may also be temporallyisolated, meaning only one wavelength or power occupies the opticaltrain at a given time, but multiple different wavelengths or powers canbe swapped. Illumination times can range from 10 femto-sec to 5 sec.Source light may also be spatially separated, meaning that light of adifferent wavelength or power may enter the cassette at the same timebut at different locations. Light sources may also be spatially andtemporally separated. The source may be capable of emitting multiplewavelengths in order to accommodate different absorption properties ofvarying materials and labels. In certain embodiments, the desiredspecificity will be a single cell per microchannel. In some embodiments,electromagnetic radiation is transmitted from the source to thesubstrate through a transmissive portion in a cassette. The signals fromeach microchannel are scanned to locate the microchannels of interest.In a one embodiment, a microchannel is screened by detecting anelectromagnetic signal emitted from a label in each cavity.

The target particles may be identified by a unique emission profile. Insome embodiments, illuminating a microchannel with a plurality ofdifferent wavelengths and detecting an emission from the microchannelcorresponds with emissions from one or more target particles. In anotherembodiment, illuminating a microchannel with a single wavelength anddetecting a plurality of emissions from the microchannel correspondswith emissions from one or more target particles. In another embodiment,illuminating a microchannel with a plurality of wavelengths anddetecting a plurality of emissions from the microchannel correspondswith emissions from one or more target particles.

Upon detecting the particle of interest, the particle of interest may beextracted, as depicted in FIG. 4E, from the substrate 130. Individualmicrochannels containing the particle of interest can be extracted usinga variety of methods. In one embodiment, the method includes pressureejection. For example, a substrate is covered by a plastic film. Themethod further provides a laser capable of making a hole through theplastic film, thereby exposing the spatially addressed microchannel.Subsequently, exposure to pressure source (e.g., air pressure) expelsthe contents from the spatially addressed microchannel. In anotherembodiment, the method of extraction involves focusing electromagneticradiation at the microchannel of the substrate to be absorbed by opaquematerial. The energy of incident radiation converts into the heat ofvaporization of a portion of the aqueous solution to generate anexpansion of the target particles or evaporation that expels at leastpart of the target particles from the microchannel of the substrate.

In certain embodiments, extraction from the microchannels of thesubstrate is accomplished by excitation of one or more particles, e.g.,opaque particles, in the microchannels of the substrate, whereinexcitation energy is focused on the particles. Accordingly, someembodiments employ energy absorbing particles in the cavities and anelectromagnetic radiation source capable of delivering electromagneticradiation of the particles in each microchannel of the substrate.Excitation of the particles in a microchannel may result in a release ofenergy that disrupts and releases the solution or mixture from themicrochannel. In certain embodiments, energy is transferred to theparticles with minimal or no increase in the temperature of the solutionor mixture within the microchannel. In certain aspects, a sequence ofpulses repeatedly agitates magnetic beads in a microchannel to disrupt ameniscus, which expels target cellular material from the substrate. Incertain aspects, the extracted cellular material is expelled onto theinternal surface of the bottom cover.

The target particles are extracted from the microchannel with a specificwavelength (third wavelength) that may be selected wavelengths rangingfrom 200 nm to about 1.5 mm. In some embodiments, the target particlesare extracted from the microchannel with a specific wavelength (thirdwavelength) that may be selected wavelengths ranging from about 350 nmto about 1200 nm. The specific wavelength for extraction may be the sameor different from the wavelengths used for illuminating and detectingthe target particles. The first, second, and third wavelengths referredto herein are independently selected from wavelengths ranging from about200 nm to about 1.5 mm. In addition, the electromagnetic radiationsource may be the same or different from the source used forilluminating and detecting the target particles. The source may becapable of emitting multiple wavelengths in order to accommodatedifferent absorption spectra of varying materials and labels. Inaddition, the electromagnetic radiation source used for illuminating,detecting, and extraction may be the same or different from each other.

As previously discussed, the extracted target particles may collect onthe internal surface of the bottom cover 120. As depicted in FIG. 4F, asolution may be added to the cassette 100 through a fill port located onthe top cover. In another embodiment, an aqueous solution may be addedto the cassette 100 through a fill port located on the bottom cover. Inone embodiment, the solution is buffer. In one embodiment, buffer may beadded to the accessory material port 112 located on the top cover 110.As depicted in FIG. 4F, the buffer may move the extracted targetparticles from the internal surface of the bottom cover to thecollection well 122. Additionally, as depicted in FIG. 4G, the extractedtarget particles may be removed from the cassette 100 through thereceiving port 123. The extracted target particles may be retrieved intoa previously sterilized container. The extracted target particles may beretrieved under sterile conditions. The extracted target particles maybe free from contaminants.

Pharmaceutical Compositions and Preparations Thereof

In some aspects, the devices and methods of the disclosure enable thepreparation of pharmaceutical compositions of cells, e.g., hematopoieticstem cells (HSCs) and/or hematopoietic stem progenitor cells (HSPCs),with unprecedented sterility, purity, and viability. The pharmaceuticalcompositions of the present disclosure may be prepared by screeningcellular material and extracting cells with desired phenotype orphenotypes.

In particular, the disclosure provides methods of sorting cellularmaterial, e.g., cells obtained from a subject. In some embodiments, themethods comprise screening cellular material, e.g., with the scannersystems and methods described herein, at a rate of approximately 100,000cells per second or more. The methods may comprise screening cellularmaterial to identify cells with a desired phenotype at a rate ofapproximately 150,000 cells per second or more, approximately 200,000cells per second or more, approximately 250,000 cells per second ormore, approximately 300,000 cells per second or more, approximately350,000 cells per second or more, approximately 400,000 cells per secondor more, approximately 450,000 cells per second or more, approximately500,000 cells per second or more, approximately 550,000 cells per secondor more, approximately 600,000 cells per second or more, approximately650,000 cells per second or more, approximately 700,000 cells per secondor more, approximately 750,000 cells per second or more, approximately800,000 cells per second or more, approximately 850,000 cells per secondor more, approximately 900,000 cells per second or more, orapproximately 950,000 cells per second or more. In certain embodiments,the methods comprise screening cellular material to identify cells of adesired phenotype at a rate of approximately 1,000.000 cells per secondor more, approximately 1,500,000 cells per second or more, approximately2,000,000 cells per second or more, approximately 2,500,000 cells persecond or more, approximately 3,000,000 cells per second or more,approximately 3,500,000 cells per second or more, approximately4,000,000 cells per second or more, approximately 4,500,000 cells persecond or more, or approximately 5,000,000 cells per second or more. Incertain embodiments, the methods comprise screening cellular material toidentify cells of a desired phenotype at a rate of approximately 100,000cells per second to about 2,000,000 cells per second.

The present disclosure provides methods for screening cellar material,e.g., with the scanner systems and methods described herein, to identifyHSCs and/or HSPCs with a desired phenotype. In some embodiments, themethods comprise screening cellular material wherein less than 10% ofthe original cellular material comprises HSCs and/or HSPCs, such as lessthan 9%, less than 8%, less than 7%, less than 6%, less than 5%, less4%, less than 3%, less than 2% or even less than 1% of the originalcellular material comprises HSCs and/or HSPCs.

In some aspects, the present disclosure provides methods for extractingcells from the original cellular material, e.g., cells obtained from ahuman subject, wherein the extracted cells are of a desired phenotype ata rate of 100,000 cells per second or more. The methods may compriseextracting cells of a desired phenotype or phenotypes from the cellularmaterial at a rate of approximately 150,000 cells per second or more,approximately 200,000 cells per second or more, approximately 250,000cells per second or more, approximately 300,000 cells per second ormore, approximately 350,000 cells per second or more, approximately400,000 cells per second or more, approximately 450,000 cells per secondor more, approximately 500,000 cells per second or more, approximately550,000 cells per second or more, approximately 600,000 cells per secondor more, approximately 650,000 cells per second or more, approximately700,000 cells per second or more, approximately 750,000 cells per secondor more, approximately 800,000 cells per second or more, approximately850,000 cells per second or more, approximately 900,000 cells per secondor more, or approximately 950,000 cells per second or more. In certainembodiments, the methods comprise extracting cells of a desiredphenotype or phenotypes at a rate of approximately 1,000,000 cells persecond or more, approximately 1,500,000 cells per second or more,approximately 2,000,000 cells per second or more, approximately2,500,000 cells per second or more, approximately 3,000,000 cells persecond or more, approximately 3,500,000 cells per second or more,approximately 4,000,000 cells per second or more, approximately4,500,000 cells per second or more, or approximately 5,000,000 cells persecond or more. The methods may comprise extracting cells of a desiredphenotype or phenotypes from the cellular material at a rate ofapproximately 150,000 cells per second to about 2,000,000 cells persecond. In certain embodiments, greater than 90%, greater than 92%,greater than 95%, greater than 98%, or greater than 99% of the extractedcells are HSCs and/or HSPCs.

In some embodiments, the methods comprise extracting cells of a desiredphenotype wherein the resulting extract has very high purity for saiddesired phenotype. Extracting cells may produce a cell extract whereinapproximately 95% or more of the cell extract are cells of a desiredphenotype. The methods may comprise extracting cells whereinapproximately 96% or more of the cell extract, approximately 97% or moreof the cell extract, approximately 98% or more of the cell extract,approximately 99% or more of the cell extract, are cells of a desiredphenotype. The extracted cells, such as extracted HSCs and/or HSPCs, maycomprise cells with a desired phenotype or phenotypes, e.g., 95% or moreof the cell extract has the desired phenotype or phenotypes. In certainembodiments, 96% or more of the cell extract, 97% or more of the cellextract, 98% or more of the cell extract, or 99% or more of the cellextract are cells of the desired phenotype or phenotypes.

In some embodiments, the methods comprise extracting cells of a desiredphenotype, wherein the cell extract has high viability. Viability of thecells may be measure in terms of cell survival subsequent to extractingthe cells. Subsequent to extracting the cells may include measuring thesurvival of the cells approximately 1 minute after to about 5 hoursafter extracting the cells. In some embodiments, the methods compriseextracting cells thereby producing a cell extract wherein the cellextract has a viability of approximately greater than 95%. In certainembodiments, the cell extract has a viability of greater than 96%,greater than 97′, greater than 98%, greater than 99%. The methods of thepresent disclosure may result in extracting cells of a desiredphenotype, wherein the cell extract has sterility suitable fortherapeutic use without the need for additional sterilizationprocedures. The cell extract may be essentially free of pathogens andother contaminants, e.g., the cell extract has less than 1% pathogens,less than 0.05% of pathogens, less than 0.01% of pathogens, or less than0.005% of pathogens. In certain embodiments, methods of the disclosureenable the preparation of cell extracts with improved therapeuticproperties, e.g., negligible graft versus host disease.

In some aspects, the present disclosure provides pharmaceuticalcompositions comprising cell extracts with one or more of the followingcharacteristics: (a) greater than 90%, greater than 95%, or greater than99% of the cells of the cell extract are HSCs and/or HSPCs; (b) the cellextract is essentially free of pathogens, e.g., less than 1%, less than0.5%, less than 0.05%, or less than 0.01% of pathogens; (c) the cellextract has a purity for cells of the desired phenotypes of greater than95%, greater than 96%, greater than 97%, greater than 98%, or greaterthan 99% of the cells in the extract; (d) the extract is suitable fortherapeutic use without additional sterilization procedures; and (e)greater than 90% of the cell extract is viable as determined followingextraction, e.g., viability measured within 2 hours of extraction.

The present disclosure provides a therapeutic composition comprisingcells, wherein greater than 95% of the cells of the compositions areHSCs and/or HSPCs, and greater than 95% of the cells are of a desiredphenotype or phenotypes. The present disclosure provides a therapeuticcomposition comprising cells, wherein greater than 95% of the cells ofthe compositions are HSCs and/or HSPCs, greater than 95% of the cellsare of a desired phenotype or phenotypes, and the composition comprisesa negligible amount of pathogens, e.g., less than 0.1%, less than 0.05%,or less than 0.001% of pathogens. The present disclosure provides atherapeutic composition comprising cells, wherein greater than 95% ofthe cells of the compositions are HSCs and/or HSPCs, greater than 95% ofthe cells are of a desired phenotype or phenotypes, and less than0.009%, less than 0.008%, less than 0.007%, less than 0.006%, less than0.005%, less than 0.004%, less than 0.003%, less than 0.002%, or lessthan 0.001% of the cells are naïve T cells. The present disclosureprovides a therapeutic composition comprising cells, wherein greaterthan 95% of the cells of the compositions are HSCs and or HSPCs, greaterthan 95% of the cells are of a desired phenotype, and 96% or more, 97%or more, 98% or more, or 99% or more of the cells are viable.

Kits

In another aspect, the present disclosure is directed to kits forsorting target particles. In one embodiment, the kits include a cassettefor sorting target particles. In certain embodiments, the cassettecontains an enclosed housing unit, a transmissive portion, a substrateencapsulated in the enclosed housing unit, and one or more fill ports.In certain embodiments, the cassette is sterilized prior to the additionof a sample material mixture.

In one embodiment, the kits may also include instructional materialscontaining directions (i.e., protocols) providing for the use of thecassette for sorting target particles. While the instructional materialstypically include written or printed materials, they are not limited tosuch. Any medium capable of storing such instructions and communicatingthem to an end user is contemplated by this disclosure. Such mediainclude, but are not limited to electronic storage media (e.g., magneticdiscs, tapes, cartridges, chips), optical media (e.g., CD ROM), and thelike. Such media may include addresses to internet sites that providesuch instructional materials.

Macro-Gel Isolation

The microchannels 610 of the substrate 130 are loaded in a specific wayto maintain the integrity of the target particles. In one embodiment, asdepicted in FIG. 6, the microchannel 610 of a substrate 130 is loadedwith a particle mixture and a sample component mixture.

As depicted in FIG. 6, the particle mixture may contain a heterogeneousmixture of opaque particles 620 and a transparent solution 630. In someembodiments, the transparent solution separates the opaque particlesfrom the sample component. In one embodiment, the opaque particles andthe sample component are separated by an average distance of 1 μm ormore. The transparent solution provides protection to the samplecomponent from energy released from the opaque particles. The opaqueparticles at least partially absorb electromagnetic radiation and atleast partially transfer the energy to the transparent solution. In someembodiments, the transfer of energy to the transparent solution causesvaporization of the surrounding transparent solution. In one embodiment,the opaque particles are not in contact with the sample component. Insome embodiments, the transparent solution is also a viscous solution.For example, the transparent solution may be agarose, collagen,matrigel, or alginate. In some embodiments, the opaque particles mayhave light-scattering properties. The opaque particles may protect thesample component from electromagnetic radiation during extraction. Theopaque particles may absorb electromagnetic radiation a specificwavelength. For example, the specific wavelengths include wavelengthsranging from about 200 nm to about 1.5 mm. As depicted in FIG. 6, thesample component mixture contains a sample component 650 and an aqueoussolution 640. The sample component may comprise a cell. The aqueoussolution may include water, buffer, media, and serum. In someembodiments, the particles mixture and the sample component mixture arein separate layers.

In various embodiments, particles mixture is cast in the microchannelprior to the addition of the sample component mixture. The particlesmixture may solidify prior to the addition of the sample componentmixture. In some embodiments, the particles mixture is added tomicrochannel of the substrate, and then the sample component mixture issubsequently added to the microchannel of the substrate. In certainembodiments, an etching solution is added to the microchannel prior tothe addition of the sample component mixture. The etching solution mayat least partially dissolve the particles mixture. The etching solutionmay include hydrofluoric acid, a strong acid such as hydrochloric acid,nitric acid, or sulfuric acid, a strong base such as sodium hydroxide orpotassium hydroxide, or any other etchant as known to one having skillin the art. The sample component remains viable after scanning andextracting with electromagnetic radiation.

Micro-Gel Isolation

In some embodiments, as depicted in FIG. 7B, sample material andparticles containing a shell 710 are simultaneously loaded into themicrochannels of the substrate. The particles may comprise an opaquecore. An opaque core may comprise of a magnetic bead or a non-magneticbead. In one embodiment, the opaque core is a magnetic bead. In anotheraspect, the shell comprises a transparent material. The transparentmaterial may be agarose, collagen, extracellular matrix, alginate, fluidfilled biological membranes, micelle, fluid filled lipid, or fatty acidvesicles. In certain embodiments, the particles not containing a shellmay be about 20 nm to about 200 μm in diameter. In certain embodiments,the particles containing a shell may be about 520 nm to about 400 μm indiameter. The sample material may be a cell. In these embodiments, thesample material may maintain viability after scanning and extractingwith electromagnetic radiation. In some embodiments, the sample materialis at least 10% viable. For example, the cells loaded with particlescontaining a shell were extracted with above 90% extraction efficiency,as depicted in FIG. 7C, and 100% cell survival, as depicted in FIG. 7D.

In another embodiment, sample material and particles containing a shellare sequentially loaded into the microchannels of the substrate. In oneembodiment, the sample material is first loaded and then, the particlescontaining the shell are load. The particles may comprise an opaquecore. An opaque core may comprise of a magnetic bead or a non-magneticbead. In one embodiment, the opaque core is a non-magnetic bead. Inanother aspect, the shell comprises a transparent material. Thetransparent material may be agarose, collagen, extracellular matrix,alginate, fluid filled biological membranes, micelle, fluid filledlipid, or fatty acid vesicles. In certain embodiments, the particles notcontaining a shell may be about 20 nm to about 200 μm in diameter. Incertain embodiments, the particles containing a shell may be about 520nm to about 400 μm in diameter. The sample material may be a cell. Inthese embodiments, the cellular material may maintain viability afterscanning and extracting with electromagnetic radiation. In someembodiments, the sample material is at least 10% viable.

In contrast, sample material loaded with particles without a shell maynot maintain viability. In some embodiments, viability of the samplematerial loaded with particles without a shell may be about 9% or lower.For example, for cells that were loaded with particles without a shell,as depicted in FIG. 7A, although the extraction efficiency was 100%, asshown in FIG. 7C, the cell survival rate was 0%, as shown in FIG. 7D.

In-Pore Spacer

The microchannels 610 of the substrate 130 are loaded in a specific wayto maintain the integrity of the sample component. In some embodiments,as depicted in FIG. 8A, the sample component may be loaded with magneticparticles 710 and non-magnetic particles 810. In some embodiments, themixture consists of magnetic particles 710 and the non-magneticparticles at a weight ratio of about 1:0.5 to 1:10. In one embodiment,the magnetic particles in the microchannel are in a concentration ofabout 1 mg/mL or about 30 mg/mL. The non-magnetic particles in themicrochannel are in a concentration of about 1 mg/mL to about 100 mg/mL.In some embodiments, the sample component is an intact or lysed cell.The non-magnetic particles 810 comprise a material that does not damagecells when excited with electromagnetic radiation. For example, thenon-magnetic particles may comprise silica, plastic, agarose, oralginate. In some embodiments, a magnetic force is applied above themicrochannel to attract the magnetic particles 710 to form a layer abovethe non-magnetic particles 810, as depicted in FIG. 8A. The magneticparticles 710 are located above the non-magnetic particles 810. Inanother embodiment, a magnetic force is applied below to microchannel toattract the magnetic particles to form a layer below the non-magneticparticles. The magnetic particles 710 are separated from the samplematerial, by at least 1 μm or more.

The ratio of magnetic particles 710 to non-magnetic particles 810 playsa role in extraction efficiency and cell viability of the cellularmaterial. For example, as depicted in FIG. 8B, when the magneticparticles 710 are held constant at 15 mg/mL, the extraction efficiencyand cell survival rate varied depending on the concentration ofnon-magnetic particles. In particular, at 5 mg/mL of non-magneticparticles, the extraction efficiency was about 57% and cell survival was0%; at 10 mg/mL of non-magnetic particles, the extraction efficiency wasabout 100% and cell survival was 0%, at 20 mg/mL of non-magneticparticles, the extraction efficiency was about 73% and cell survival wasabout 63%; and at 40 mg/mL of non-magnetic particles, the extractionefficiency was about 80% and cell survival was about 25%.

In another aspect, the sample component may be loaded with opaqueparticles in a sequential manner. First, the sample component and aplurality of opaque particles that do not absorb a specific wavelengthare loaded to the microchannel of the substrate. The specificwavelengths include wavelengths ranging from about 200 nm to about 1.5mm. Then, opaque particles which absorb a specific wavelength are loadedto the microchannel. In one embodiment, the opaque particles comprise ofmagnetic and non-magnetic particles. In one embodiment, the opaqueparticles comprise of magnetic particles.

Sequential Loading

In various embodiments of a method of quantifying the number of samplematerial in a microchannel of a substrate, the microchannels 610 of thesubstrate 130 may be sequentially loaded to enhance sample visibility.In certain embodiments, sample material may comprise a cell. In anexemplary embodiment, as depicted in FIG. 9A, the sample mixture isfirst added to the microchannels of the substrate, and then, theparticles are subsequently added to the microchannels of the substrate.Then, the number of sample material in the microchannels may bequantified. In various embodiments, the particles are opaque beads. Forexample, opaque beads include Dynabead®, agarose, and ProMag®. In someembodiments, the particles are added to the microchannels of thesubstrate after about 5 mins or more after the addition of samplemixture. In certain embodiments, the particles are added to themicrochannels of the substrate after about 10 mins after the addition ofsample mixture. In one embodiment, the amount of sample material may bequantified by microscopy. In certain embodiments, the amount of cellsmay be quantified by microscopy. In some embodiments, such sequentialloading prevent the cells from being obscured from view by the beads.For example, as depicted in FIG. 9B to 9G, three types of particles(i.e., Dynabead®, agarose, and ProMag®) were applied to themicrochannels of a substrate in both a sequential manner and in a mixedbatch with cellular material. Dynabead® was used in FIGS. 9B and 9C,agarose was used in FIGS. 9D and 9E, and ProMag® was used in FIGS. 9Fand 9G. Additionally, FIG. 9H shows a graphical representation of thecontrol that contained no particles. The accuracy of the cell count ineach microchannel utilizing the sequential loading method and the mixedbatch method was compared to the control in FIG. 9H. As shown in FIG. 9Bto 9G, the microchannels that were loaded with the sequential loadingmethod (FIGS. 9C, 9E, and 9G) more closely resembled the cell count inthe control (FIG. 9H) than the microchannels that were loaded with themixed batch method (FIGS. 9B, 9D, and 9F).

Fluorescence Displacement

In certain embodiments, the microchannels 610 of the substrate 130 areloaded with fluorescent material to enhance sample visibility. In anexemplary embodiment, as depicted in FIG. 10, sample material andfluorescent material are added to the microchannels, and later thesample material is quantified. In certain embodiments, sample materialmay comprise a cellular material. In certain embodiments, cellularmaterial may comprise cells. Each microchannel of the substrate 130 isloaded near uniformly with fluorescent material. Fluorescent materialmay be in solution or attached to a solid, such as opaque beads. Forexample, fluorescent material may include fluorescein isothiocyanate,(FITC), Alexa Fluor®, Alexa Fluor® 594, Alexa Fluor® 647, Alexa Fluor®700, Brilliant Violet™ 421, R-phycoerythrin (PE), PE-Cy® 5,allophycocyanin (APC), PE Texas Red®, green fluorescent protein (GFP),and yellow fluorescent protein (YFP). The presence of one or more cellslowers the fluorescent signal from the microchannel. The presence of acell reduces the fluorescent intensity by at least 5% of the max valueand no more than 95% of the max value. In this embodiment, highfluorescent intensity in the microchannel correlates with no cell countor a low cell count in the microchannel. Low fluorescent intensitycorrelates with a high cell count in the microchannel. For example, asseen in FIGS. 10C and 10D, the microchannels of the substrate that areloaded with fluorescent material and do not contain cells, thosecorresponding regions are brighter than the regions that contain cells.The regions that contain cells are circled in FIG. 10D. Additionally,FIGS. 10E and 10F show the regions that contain cells through the use ofAPC cells and its corresponding bright regions. FIG. 10G shows that theaverage fluorescent intensity of a microchannel with no cell is greaterthan the average fluorescent intensity of a microchannel with a cell.

Ultrafast Sorter

In one embodiment of an optical apparatus for laser scanning cellsorting, FIG. 11 illustrates an optical apparatus utilizing a rotatingpolygon mirror. The apparatus 1100 comprises a first fluorescenceexcitation light source 1110 and a second fluorescence excitation lightsource 1112. In some cases, the apparatus may comprise a plurality offluorescence excitation light sources. In some cases, the plurality ofexcitation light sources may overlap. In some cases, the plurality ofexcitation light sources may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or greater than 12 fluorescence excitation light sources. One ormore of the plurality of fluorescence excitation light sources maycomprise laser light sources. One or more of the excitation lightsources may comprise light emitting diode (LED) light sources. In somecases, one or more of the plurality of fluorescence excitation lightsources may emit light at a wavelength corresponding to the fluorescenceexcitation wavelength of a particular fluorophore. In some cases, one ormore of the excitation light sources may be configured to emit aplurality of wavelengths of light. In some cases, one or more of theexcitation light sources may be configured to emit a plurality ofwavelengths of light, such that each of the plurality of wavelengthscomprises a peak separated from others peaks of the plurality ofwavelengths. One or more of the plurality of fluorescence excitationlight sources may emit light at a wavelength corresponding to anexcitation wavelength of a fluorophore that is endogenous to a cell toexcite autofluorescence. For instance, one or more of the plurality offluorescence excitation light sources may be tuned to the excitationwavelength of an autofluorescent molecule such as nicotinamide adeninedinucleotide phosphate (NADPH), chlorophyll, collagen, retinol,riboflavin, cholecalciferol, folic acid, pyridoxine, tyrosine,dityrosine, indolamine, lipofuscin, polyphenol, tryptophan, flavin, ormelanin. One or more of the excitation light sources may emit light at awavelength corresponding to an excitation wavelength of anyautofluorescent molecule.

One or more of the plurality of fluorescence excitation light sourcesmay emit light at a wavelength corresponding to an excitation wavelengthof a fluorophore that is exogenous to a cell. For instance, one or moreof the plurality of fluorescence excitation lights sources may be tunedto the excitation wavelength of hydroxycoumarin, methoxycoumarin, Alexafluor, aminocoumarin, Cy2, carboxyfluorescein (FAM), Alexa fluor 488,fluorescein isothiocyanate (FITC), Alexa fluor 430, Alexa fluor 532,6-carboxy-2,4,4,5,7,7-hexachlorofluorescein (HEX), Cy3,tetramethylrhodamine (TRITC), Alexa fluor 546, Alexa fluor 555,R-phycoerythrin, Rhodamine Red-X, Tamara, Cy3.5 581, Rox, Alexa fluor568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633,allophycocyanin, Alexa fluor 647, Cy5, Alexa fluor 660, Cy5.5, TruRed,Alexa fluor 680, Cy7, or any other fluorophore as known to one havingskill in the art.

Light from the fluorescence excitation light sources is directed to adichroic mirror 1120 and passed to a beam expander 1122. In some cases,a plurality of dichroic mirrors are utilized to direct light from morethan two excitation light sources to the beam expander. The apparatusmay comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12dichroic mirrors. Upon expansion of the beams in the beam expander,light from the fluorescence excitation light sources is then directed toa mirror 1124. In some cases, the mirror 1124 comprises a galvanometer.The mirror directs the light to a rotating polygon mirror 1140. In somecases, the polygon mirror may be replaced by a resonant scanning mirror.

In certain embodiments, the polygon mirror may be substituted with adigital light processing system (DLPS). The DLPS may comprise aplurality of independently addressable micromirrors. The DLPS may beirradiated with a light source. Each of the plurality of micromirrorsmay be independently positioned to either direct light to a particularlocation or to prevent light from reaching a particular location. Eachmicromirror may be addressed electronically.

The rotating polygon mirror rapidly scans light from the fluorescenceexcitation light sources along a curved focal plane perpendicular to theaxis of rotation of the polygon mirror. An F-theta lens 1150 produces afocal plane that is substantially flat. After reflection from a surfaceof the rotating polygon mirror and refraction through the F-theta lens,light from the excitation light sources is directed to the cassette 100.The F-theta lens may be configured to focus the plurality of excitationbeams to a microchannel in the cassette. As the rotating polygon mirrorrotates, light from the excitation light sources is scanned across aline of microchannels in the cassette. After a scan across a singleline, the cassette may be moved to allow scanning of a new line. In somecases, the movement is timed to the scanning of a single line. Thetiming may be accomplished, for instance, by coordinating the movementof the cassette and the scanning of light through a synchronized clocksignal.

Upon receiving light from the excitation light sources, one or morecells in a microchannel may fluoresce and emit light of a greaterwavelength than the wavelength of the excitation light source thatstimulated the fluorescence. The fluorescence may be due to the presenceof endogenous fluorophores located within or on the cell. Thefluorescence may be due to the presence of exogenous fluorophores. Theemitted light is received and guided by a light guide 1160. The lightguide may comprise an optical fiber. The light guide may comprise anoptical fiber bundle. The light guide may comprise one or more mirrors.The one or more mirrors may comprise flat mirrors. The one or moremirrors may comprise flat dichroic mirrors. The one or more mirrors maycomprise concave mirrors. The one or more mirrors may comprise sphericalmirrors. The emitted light is directed to a set of coupling optics 1162and to a beamsplitter 1164. The beamsplitter allows one wavelength ofemitted light to pass to a first detector 1172 and redirects anotherwavelength of emitted light to a second detector 1174. In some cases,the apparatus may comprise a plurality of beamsplitters. In some cases,the apparatus may comprise a plurality of detectors. In some cases, theplurality of beamsplitters may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or greater than 12 beamsplitters. In some cases, the pluralityof detectors may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, orgreater than 12 detectors. Each beamsplitter of the plurality ofbeamsplitters may allow one or more wavelengths of emitted light to passand may redirect other wavelengths of emitted light. Each detector ofthe plurality of detectors may be a photodiode, a photomultiplier tube,or any other optical detector as known to one having skill in the art.

Each detector registers a signal corresponding to the intensity ofemitted light having a particular wavelength. The light intensity ateach detector is electronically sampled, digitized, and directed toelectronic circuitry (not shown). The electronic circuitry processes thesignals from each detector in order to make a determination as towhether a particular microchannel contains a cell that should be removedfrom the microchannel. In some cases, the electronic circuitry acts todetermine whether the intensities of emitted light detected by each ofthe plurality of detectors exceeds a threshold value. In some cases, theelectronic circuitry acts to determine whether the intensities ofemitted light detected by each of the plurality of detectors fallswithin a range of values. In some cases, the range of values may bedifferent for each detector. If the intensities of emitted lightdetected by each of the plurality of detectors lies within the range ofvalues, the electronic circuitry sends a signal to remove thecorresponding cell from the given microchannel. In some cases, thecircuitry is implemented as firmware. In some cases, the circuitry isimplemented as a field programmable gate array (FPGA).

In some instances, the range of values for each detector may be unknownat the outset of the scanning procedure. For instance, the range ofvalues may depend on the cell type (e.g. red blood cell, white bloodcell, etc). The range of values may depend on operating parameters ofthe system (e.g. age-related degradation of optical components). Thus,it may be beneficial to determine acceptable ranges of values uponreceiving a new cassette in the system. For instance, such ranges may bedetermined by scanning the plurality of excitation light sources over asample population of cells and determining the intensities at eachdetector for the population of cells. The population of cells maycomprise more than 1,000, more than 10,000, more than 100,000, or morethan 1,000,000 cells. The population of cells may lie in a range definedby any two of the preceding values. The range of values may be chosensuch that only a subpopulation of the cells from the sample populationgives rise to fluorescence intensities falling within the range ofvalues. For instance, the range of values for each detector may bechosen such that less than 1%, less than 5%, less than 10%, less than20%, or less than 50% of the cells give rise to fluorescence intensitiesat each detector that fall within the range of values. The range ofvalues may be chosen to lie within a range defined by any two of thepreceding values. These ranges of values may then be used to determinewhether a given cell should be removed from a given microchannel duringa scan of the entire cassette.

When the electronic circuitry makes a determination that a given cell ina given microchannel meets the criteria for removal from themicrochannel, a signal may be sent to direct light from an extractionlaser source 1130 to the microchannel. In some cases, the extractionlaser and the circuitry may be synchronized to a shared clock. Theextraction laser source may emit light at a wavelength that allowsextraction of a cell from a microchannel. For instance, the extractionlaser source may emit light at a wavelength that is absorbed by aparticle in a microchannel, as described herein. The extraction lasersource may emit light at a wavelength in the ultraviolet, visible, ornear infrared region of the electromagnetic spectrum. The extractionlaser source may emit light in a range of wavelengths from about 200 nmto about 2000 nm. The extraction laser source may comprise a continuouswave laser source. The extraction source may comprise a quasi-continuouswave laser source. The extraction laser source may comprise a pulsedlaser source. The pulsed laser source may emit laser pulses having aduration shorter than 10 fs, shorter than 100 fs, shorter than 1 μs,shorter than 10 μs, shorter than 100 μs, shorter than 1 ns, shorter than10 ns, shorter than 100 ns, shorter than 1 μs, shorter than 10 μs,shorter than 100 μs, or shorter than 1 ms. The pulsed laser source mayemit laser pulses having a duration lying in a range defined by any twoof the preceding values. In some cases, the pulsed laser source may emitlaser pulses having a duration within the range from about 1 fs to about100 μs. In some cases, the pulsed laser source may emit laser pulseshaving a duration within the range from about 100 ns to about 10 μs. Thepulsed laser source may emit laser pulses having an adjustable duration.

The pulsed laser source may emit laser pulses with a repetition rate ofless than 1 kHz, less than 10 kHz, less than 100 kHz, less than 1 MHz,less than 10 MHz, less than 100 MHz, or less than 1 GHz. The pulsedlaser source may emit laser pulses with a repetition rate lying in arange defined by any two of the preceding values. In some cases, thepulsed laser source may emit pulses with a repetition rate within therange from about 10 KHz to about 1 MHz. In some cases, each pulse mayproduce less than 1 nJ, less than 10 nJ, less than 100 nJ, less than 1μJ, less than 10 μJ, less than 100 μJ, or less than 1 mJ of energy. Eachpulse may produce an energy lying in a range defined by any two of thepreceding values. In some cases, each pulse may produce an energy lyingin the range from about 1 μJ to about 50 μJ. The pulsed laser source mayemit a peak power in the range from 0.1 W to 10⁷ W.

The pulsed laser source may be a fiber laser source. The pulsed lasersource may be a pulse-on-demand fiber laser source. The pulsed lasersource may be a master oscillator fiber amplifier (MOPA) laser source.The pulsed laser source may be a doped fiber laser source. The pulsedlaser source may be a rare earth ion doped fiber laser source. Thepulsed laser source may be a ytterbium doped fiber laser source. Thepulsed laser source may be a laser utilizing a doped crystal gainmedium. The pulsed laser source may be a laser utilizing aneodymium-doped crystal gain medium. The pulsed laser source may be aNd:YAG laser. The pulsed laser source may be a Nd:YVO₄ laser. The pulsedlaser source may be a semiconductor laser. The pulsed laser source maybe a diode laser. The pulsed laser source may be a vertical cavitysurface emitting laser (VCSEL). The pulsed laser source may be a VCSELarray. The pulsed laser source may be a gas laser. The pulsed lasersource may be a CO₂ laser. The pulsed laser source may be an excimerlaser. The pulsed laser source may employ Q-switching. The pulsed lasersource may employ mode locking. The pulsed laser source may be anypulsed laser source as is known to one having skill in the art.

The pulsed laser source may direct a laser pulse to a microchannel inresponse to a signal from electronic circuitry in response tointensities of light detector at each of the plurality of detectors. Thepulse may be directed to the microchannel using an acousto-opticalmodulator (AOM), electro-optic modular (EOM), or any modulation deviceas may be known to one having skill in the art. For instance, themodulation device may be configured to pass the zero-order diffractedbeam only in response to a signal that the extraction laser shouldremove a cell from a microchannel; at all other times, the modulationdevice may be configured to pass the first-order diffracted beam orhigher-order diffracted beam. In some cases, the pulsed laser mayproduce pulses only in response to a signal that the extraction lasershould remove a cell from a microchannel. For instance, a doped fiberlaser may be operated in a reverse-biased configuration until receivinga signal to direct a pulse to the microchannel; upon receiving thesignal to direct a pulse to the microchannel, the doped fiber laser maybe operated in the forward-biased configuration.

The extraction laser may be directed to the rotating polygon mirror by amirror 1132. The rotating polygon mirror rapidly scans light from theextraction laser along a curved focal plane perpendicular to the axis ofrotation of the polygon mirror. The F-theta lens produces a focal planethat is substantially flat. After reflection from a surface of therotating polygon mirror and refraction through the F-theta lens, lightfrom the extraction laser is directed to the cassette 100. The F-thetalens may be configured to focus the plurality of excitation beams to amicrochannel in the cassette. When the extraction laser is directed to amicrochannel, energy from the laser causes cavitation of the liquidsample in which a cell is suspended. This causes the removal of the cellfrom the microchannel.

The system may be configured to scan both the plurality of fluorescenceexcitation beams and the extraction beam. In some cases, the system maybe configured to scan the extraction beam so that it is separated fromthe plurality of fluorescence excitation beams. In some cases, thesystem may be configured to focus the plurality of excitation lightsources on a first microchannel and to simultaneously focus theextraction laser on a second microchannel. In some cases, the extractionbeam may be separated from the plurality of fluorescence excitationbeams by a distance of less than 1 μm, 5 μm, 10 μm, less than 50 μm,less than 100 μm, less than 500 μm, less than 1 mm, less than 5 mm, orless than 10 mm. In some cases, the extraction beam may be separatedfrom the plurality of fluorescence excitation beams by a distance lyingin a range defined by any two of the preceding values. In some cases,the extraction beam may be separated from the plurality of fluorescenceexcitations beams by a distance within a range from about 100 μm toabout 5 mm. In some cases, the extraction beam may be separated from theplurality of fluorescence excitations beams by a distance within a rangefrom about 100 μm to about 1 mm. In some cases, the extraction beam maybe separated from the plurality of fluorescence excitations beams by adistance within a range from about 10 μm to about 1000 mm. The pluralityof excitation light sources and the extraction beam may be located onthe same side of the cassette. The plurality of excitation light sourcesand the extraction beam may be located on opposite sides of thecassette.

In some cases, the system may be configured to scan the microchannels ata rate of more than 10,000 channels per second, more than 50,000channels per second, more than 100,000 channels per second, more than500,000 channels per second, more than 1,000,000 channels per second,more than 5,000,000 channels per second, more than 10,000,000 channelsper second, more than 50,000,000 channels per second, or more than100,000,000 channels per second. The system may be configured to scanthe microchannels at a rate lying in a range defined by any two of thepreceding values. In some cases, the system may be configured to scanthe microchannels at a rate within a range from about 3,000 to about300,000,000 channels per second.

System 1100 may scan the substrate at a rate greater than 1,000,000microchannels per second, greater than 2,000,000 microchannels persecond, or greater than 3,000,000 microchannels per second. System 1100may scan the substrate at a rate that is within a range defined by anytwo of the preceding values. System 1100 may extract target particlesfrom the substrate at a rate greater than 500,000 microchannels persecond, greater than 600,000 microchannels per second, greater than700,000 microchannels per second, greater than 800,000 microchannels persecond, greater than 900,000 microchannels per second, or greater than1,000,000 microchannels per second. System 1100 may extract targetparticles at a rate that is within a range defined by any two of thepreceding values. System 1100 may extract the target particles such thata collection of extracted target particles has a purity greater than90%, greater than 95%, or greater than 99%. System 1100 may extract thetarget particles such that the collection of extracted target particleshas a purity that is within a range defined by any two of the precedingvalues.

Though shown as forming a single device in FIG. 11, system 1100 may beconfigured such that the fluorescence subsystem (comprising elements1110, 1112, 1120, 1122, 1124, 1140, 1150, 1160, 1162, 1164, 1170, and1172) are arranged as a fluorescence device and the extraction subsystem(comprising elements 1130, 1132, 1140, and 1150) are arranged as anextraction device. In such an arrangement, the substrate 100 may besubjected to a fluorescence analysis as described herein using thefluorescence device. Following the fluorescence analysis, the substratemay be transferred to the extraction device for extraction of locationsof interest on the substrate identified during the fluorescenceanalysis. Proper alignment of the substrate in each of the fluorescencedevice and the extraction device may be achieved by referring to one ormore fiducial markers on the substrate. In some cases, arranging system1100 on two devices may require duplication of one or more elements ofsystem 1100. For instance, elements 1140 and 1150 may be required onboth the fluorescence device and the extraction device.

In another embodiment of an optical apparatus for laser scanning cellsorting, FIG. 12 illustrates an optical apparatus utilizing two rotatingpolygon mirrors. The apparatus 1200 comprises all of the elements ofapparatus 1100 from FIG. 11. The apparatus comprises: a plurality ofexcitation light sources 1110 and 1112, a dichroic mirror 1120, a beamexpander 1122, an excitation light mirror 1124, an extraction laser1130, an extraction laser mirror 1132, a rotating polygon mirror 1140,an F-theta lens 1150, a light guide 1160, coupling optics 1162, aplurality of beamsplitters 1164, and a plurality of detectors 1170 and1172.

Additionally, the apparatus 1200 comprises a second rotating polygonmirror 1142 and a second F-theta lens 1152. In some cases, the secondpolygon mirror may be replaced by a resonant scanning mirror. The systemmay be configured such that the plurality of excitation light sourcesare directed to the cassette 100 by the first rotating polygon mirror1140 and the first F-theta lens 1150, while the extraction laser isdirected to the cassette by the second rotating polygon mirror 1142 andthe second F-theta lens 1152. The plurality of excitation light sourcesand the extraction beam may be located on the same side of the cassette.The plurality of excitation light sources and the extraction beam may belocated on opposite sides of the cassette.

The first rotating polygon mirror rapidly scans light from thefluorescence excitation light sources along a curved focal planeperpendicular to the axis of rotation of the polygon mirror. The firstF-theta lens produces a focal plane that is substantially flat. Afterreflection from a surface of the first rotating polygon mirror andrefraction through the first F-theta lens, light from the excitationlight sources is directed to the cassette 100. The first F-theta lensmay be configured to focus the plurality of excitation beams to amicrochannel in the cassette. As the first rotating polygon mirrorrotates, light from the excitation light sources is scanned across aline of microchannels in the cassette.

The second rotating polygon mirror rapidly scans light from theexcitation laser along a curved focal plane perpendicular to the axis ofrotation of the polygon mirror. The second F-theta lens produces a focalplane that is substantially flat. After reflection from a surface of thesecond rotating polygon mirror and refraction through the second F-thetalens, light from the extraction laser is directed to the cassette 100.The second F-theta lens may be configured to focus the extraction laserto a microchannel in the cassette. As the second rotating polygon mirrorrotates, light from the extraction laser is scanned across a line ofmicrochannels in the cassette.

The system may be configured to scan both the plurality of fluorescenceexcitation beams, using the first rotating polygon mirror, and theextraction beam, using the second rotating polygon mirror. In somecases, the system may be configured to scan the extraction beam so thatit is separated from the plurality of fluorescence excitation beams. Insome cases, the system may be configured to focus the plurality ofexcitation light sources on a first microchannel and to simultaneouslyfocus the extraction laser on a second microchannel. In some cases, theextraction beam may be separated from the plurality of fluorescenceexcitation beams by a distance of less than 1 μm, 5 μm, 10 μm, less than50 μm, less than 100 μm, less than 500 μm, less than 1 mm, less than 5mm, or less than 10 mm. In some cases, the extraction beam may beseparated from the plurality of fluorescence excitation beams by adistance lying in a range defined by any two of the preceding values. Insome cases, the extraction beam may be separated from the plurality offluorescence excitations beams by a distance within a range from about100 μm to about 5 mm. In some cases, the extraction beam may beseparated from the plurality of fluorescence excitations beams by adistance within a range from about 100 μm to about 1 mm. In some cases,the extraction beam may be separated from the plurality of fluorescenceexcitations beams by a distance within a range from about 10 μm to about1000 mm. In some cases, the scanning of the plurality of excitationlight sources and the excitation laser is synchronized. In some cases,the synchronization is achieved through a synchronized clock signal. Theplurality of excitation light sources and the extraction beam may belocated on the same side of the cassette. The plurality of excitationlight sources and the extraction beam may be located on opposite sidesof the cassette.

In some cases, the system may be configured to scan the microchannels ata rate of more than 10,000 channels per second, more than 50,000channels per second, more than 100,000 channels per second, more than500,000 channels per second, more than 1,000,000 channels per second,more than 5,000,000 channels per second, more than 10,000,000 channelsper second, more than 50,000,000 channels per second, or more than100,000,000 channels per second. The system may be configured to scanthe microchannels at a rate lying in a range defined by any two of thepreceding values. In some cases, the system may be configured to scanthe microchannels at a rate within a range from about 3,000 to about300,000,000 channels per second.

System 1200 may scan the substrate at a rate greater than 1,000,000microchannels per second, greater than 2,000,000 microchannels persecond, or greater than 3,000,000 microchannels per second. System 1200may scan the substrate at a rate that is within a range defined by anytwo of the preceding values. System 1200 may extract target particlesfrom the substrate at a rate greater than 500,000 microchannels persecond, greater than 600,000 microchannels per second, greater than700,000 microchannels per second, greater than 800,000 microchannels persecond, greater than 900,000 microchannels per second, or greater than1,000,000 microchannels per second. System 1200 may extract targetparticles at a rate that is within a range defined by any two of thepreceding values. System 1200 may extract the target particles such thata collection of extracted target particles has a purity greater than900%, greater than 95%, or greater than 99%. System 1200 may extract thetarget particles such that the collection of extracted target particleshas a purity that is within a range defined by any two of the precedingvalues.

Though shown as forming a single device in FIG. 12, system 1200 may beconfigured such that the fluorescence subsystem (comprising elements1110, 1112, 1120, 1122, 1124, 1140, 1150, 1160, 1162, 1164, 1170, and1172) are arranged as a fluorescence device and the extraction subsystem(comprising elements 1130, 1132, 1142, and 1152) are arranged as anextraction device. In such an arrangement, the substrate 100 may besubjected to a fluorescence analysis as described herein using thefluorescence device. Following the fluorescence analysis, the substratemay be transferred to the extraction device for extraction of locationsof interest on the substrate identified during the fluorescenceanalysis. Proper alignment of the substrate in each of the fluorescencedevice and the extraction device may be achieved by referring to one ormore fiducial markers on the substrate.

In another embodiment of an optical apparatus for laser scanning cellsorting, FIG. 13 illustrates an optical apparatus utilizing two rotatingpolygon mirrors and a confocal detection technique. The apparatus 1300comprises many of the elements of apparatus 1100 from FIG. 11. Theapparatus comprises: a plurality of excitation light sources 1110 and1112, a dichroic mirror 1120, abeam expander 1122, an excitation lightmirror 1124, an extraction laser 1130, an extraction laser mirror 1132,a rotating polygon mirror 1140, an F-theta lens 1150, coupling optics1162, a plurality of beamsplitters 1164, and a plurality of detectors1170 and 1172.

Additionally, the apparatus 1300 may comprise a second rotating polygonmirror 1142 and a second F-theta lens 1152. The system may be configuredsuch that the plurality of excitation light sources are directed to thecassette 100 by the first rotating polygon mirror 1140 and the firstF-theta lens 1150, while the extraction laser is directed to thecassette by the second rotating polygon mirror 1142 and the secondF-theta lens 1152. The plurality of excitation light sources and theextraction beam may be located on the same side of the cassette. Theplurality of excitation light sources and the extraction beam may belocated on opposite sides of the cassette.

In place of the light guide, the apparatus 1300 further comprises a setof mirrors to direct light to the one or more detectors. The excitationlight mirror 1124 may be a dichroic mirror. The confocal detectioncavity may comprise mirrors 1166 and 1168. The mirrors may be flatmirrors. The mirrors may be concave mirrors. The mirrors may bespherical mirrors. The mirrors may be arranged in a confocalconfiguration.

System 1300 may scan the substrate at a rate greater than 1,000,000microchannels per second, greater than 2,000,000 microchannels persecond, or greater than 3,000,000 microchannels per second. System 1300may scan the substrate at a rate that is within a range defined by anytwo of the preceding values. System 1300 may extract target particlesfrom the substrate at a rate greater than 500,000 microchannels persecond, greater than 600,000 microchannels per second, greater than700,000 microchannels per second, greater than 800,000 microchannels persecond, greater than 900,000 microchannels per second, or greater than1,000,000 microchannels per second. System 1300 may extract targetparticles at a rate that is within a range defined by any two of thepreceding values. System 1300 may extract the target particles such thata collection of extracted target particles has a purity greater than900%, greater than 95%, or greater than 99%. System 1300 may extract thetarget particles such that the collection of extracted target particleshas a purity that is within a range defined by any two of the precedingvalues.

Though shown as forming a single device in FIG. 13, system 1300 may beconfigured such that the fluorescence subsystem (comprising elements1110, 1112, 1120, 1122, 1124, 1140, 1150, 1164, 1166, 1168, 1170, and1172) are arranged as a fluorescence device and the extraction subsystem(comprising elements 1130, 1132, 1142, and 1152) are arranged as anextraction device. In such an arrangement, the substrate 100 may besubjected to a fluorescence analysis as described herein using thefluorescence device. Following the fluorescence analysis, the substratemay be transferred to the extraction device for extraction of locationsof interest on the substrate identified during the fluorescenceanalysis. Proper alignment of the substrate in each of the fluorescencedevice and the extraction device may be achieved by referring to one ormore fiducial markers on the substrate.

In another embodiment of an optical apparatus for laser scanning cellsorting, FIG. 14 illustrates an optical apparatus utilizing agalvanometer scanning mechanism. The apparatus 1400 comprises afluorescence excitation light source 1410. In some cases, the apparatusmay comprise a plurality of fluorescence excitation light sources. Insome cases, the plurality of excitation light sources may overlap. Insome cases, the plurality of excitation light sources may comprise 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater than 12 fluorescenceexcitation light sources. One or more of the plurality of fluorescenceexcitation light sources may comprise laser light sources. One or moreof the excitation light sources may comprise light emitting diode (LED)light sources. In some cases, one or more of the plurality offluorescence excitation light sources may emit light at a wavelengthcorresponding to the fluorescence excitation wavelength of a particularfluorophore. In some cases, one or more of the excitation light sourcesmay be configured to emit a plurality of wavelengths of light. In somecases, one or more of the excitation light sources may be configured toemit a plurality of wavelengths of light, such that each of theplurality of wavelengths comprises a peak separated from others peaks ofthe plurality of wavelengths. One or more of the plurality offluorescence excitation light sources may emit light at a wavelengthcorresponding to an excitation wavelength of a fluorophore that isendogenous to a cell to excite autofluorescence. For instance, one ormore of the plurality of fluorescence excitation light sources may betuned to the excitation wavelength of an autofluorescent molecule suchas nicotinamide adenine dinucleotide phosphate (NADPH), chlorophyll,collagen, retinol, riboflavin, cholecalciferol, folic acid, pyridoxine,tyrosine, dityrosine, indolamine, lipofuscin, polyphenol, tryptophan,flavin, or melanin. One or more of the excitation light sources may emitlight at a wavelength corresponding to an excitation wavelength of anyautofluorescent molecule.

One or more of the plurality of fluorescence excitation light sourcesmay emit light at a wavelength corresponding to an excitation wavelengthof a fluorophore that is exogenous to a cell. For instance, one or moreof the plurality of fluorescence excitation lights sources may be tunedto the excitation wavelength of hydroxycoumarin, methoxycoumarin, Alexafluor, aminocoumarin, Cy2, carboxyfluorescein (FAM), Alexa fluor 488,fluorescein isothiocyanate (FITC), Alexa fluor 430, Alex fluor 532,6-carboxy-2,4,4,5,7,7-hexachlorofluorescein (HEX), Cy3,tetramethylrhodamine (TRITC), Alexa fluor 546, Alexa fluor 555,R-phycoerythrin, Rhodamine Red-X, Tamara, Cy3.5 581, Rox, Alexa fluor568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633,allophycocyanin, Alexa fluor 647, Cy5, Alexa fluor 660, Cy5.5, TruRed,Alexa fluor 680, Cy7, or any other fluorophore as known to one havingskill in the art.

Light from the fluorescence excitation light sources is directed to aset of conditioning optics. The conditioning optics may comprise a firstlens 1420 and a second lens 1424. The two lenses may act to expand thebeamwaist of the excitation light, reduce the beamwaist of theexcitation light, and/or to collimate the excitation light. Theconditioning optics may further comprise a filter wheel 1422.

The excitation light is then passed to a dichroic mirror 1480. In somecases, a plurality of dichroic mirrors are utilized to direct light frommore than two excitation light sources to the beam expander. Theapparatus may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or morethan 12 dichroic mirrors. In some cases, the apparatus may comprise amulti-edge dichroic cube.

The excitation light is directed to a dichroic mirror 1460. The dichroicmirror 1460 may be a dichroic mirror configured to pass light having awavelength in the ultraviolet or visible region of the electromagneticspectrum and to reflect light having a wavelength in the infrared regionof the electromagnetic spectrum. The excitation light is directed to anobjective lens 1470. The objective lens may have a magnification greaterthan 1×, 2×, 5×, 10×, 20×, 50×, 100×, 200×, 500×, 1000×. The objectivelens may have a magnification in a range defined by any two of thepreceding values. The objective lens may be an infinity correctedobjective lens. The objective lens provides a large excitation field anda large field-of-view. This allows the excitation light source toilluminate a large area of the cassette 100 without requiring scanningof the excitation light sources. In some cases, the apparatus maycomprise a plurality of objective lenses having a plurality offields-of-view.

In some cases, the apparatus is configured to transmit the excitationlight coaxially with the field-of-view through the objective lens. Insome cases, the excitation light comprises diffused excitation light. Insome cases, the excitation light comprises diffused infinity correctedexcitation light. The excitation light may excite fluorescence over anexcitation field of less than 1 mm, less than 5 mm, less than 10 mm,less than 50 mm, or less than 100 mm. The excitation light may excitefluorescence over an excitation field lying in a range defined by anytwo of the preceding values. The objective lens may have a field-of-viewof less than 1 mm, less than 5 mm, less than 10 mm, less than 50 mm, orless than 100 mm. The objective lens may have a field-of-view lying in arange defined by any two of the preceding values. In some cases, thefield-of-view is defined with an optical structure. The opticalstructure may be an aperture. The optical structure may be a dimensionacross an aperture. The optical structure may be a pinhole. The opticalstructure may be a mirror. The optical structure may be a dimensionacross a reflective surface of a mirror.

Upon receiving light from the excitation light sources, one or morecells in a microchannel may fluoresce and emit light of a greaterwavelength than the wavelength of the excitation light source thatstimulated the fluorescence. The fluorescence may be due to the presenceof endogenous fluorophores located within or on the cell. Thefluorescence may be due to the presence of exogenous fluorophores. Theemitted light is directed to a wavelength selector 1482 to selectdesired wavelengths of light. In some cases, the wavelength selectorproduces filtered light. The filtered light is directed to atwo-dimensional array detector 1490 using a lens 1484. In some cases,the two-dimensional array detector comprises a camera. In some cases,the lens 1484 comprises a tube lens. The camera produces a fluorescenceimage of cells that are located within its field-of-view.

The wavelength selector may comprise a filter. The filter may comprisean emission filter. The filter may comprise an emission filter wheel.The emission filter wheel may comprise a plurality of filters. In somecases, the apparatus may comprise a plurality of wavelength-selectivefilters. The wavelength selector may comprise a dichroic mirror. Thewavelength selector may comprise a prism. The wavelength selector maycomprise a diffraction grating. The apparatus may comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, or greater than 12 wavelength selectors. Insome cases, the apparatus may comprise a plurality of cameras. Theapparatus may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greaterthan 12 cameras. Each filter of the plurality of wavelength selectorsmay allow one or more wavelengths of emitted light to pass to one ormore of the plurality of cameras and may redirect or absorb otherwavelengths of emitted light. Each camera of the plurality of camerasmay be a charge-coupled device (CCD) camera, a complementary metal oxidesemiconductor (CMOS) camera, or any other camera as known to one havingskill in the art.

Each camera registers an image corresponding to the intensity of emittedlight having a particular wavelength. The light intensity at each camerais electronically sampled, digitized, and directed to electroniccircuitry (not shown). The electronic circuitry processes the signalsfrom each camera in order to make a determination as to whether aparticular microchannel contains a cell that should be removed from themicrochannel. In some cases, the electronic circuitry acts to determinewhether the intensities of emitted light at each pixel detected by eachof the plurality of cameras exceeds a threshold value. In some cases,the electronic circuitry acts to determine whether the intensities ofemitted light at each pixel detected by each of the plurality of camerasfalls within a range of values. In some cases, the range of values maybe different for each camera. If the intensities of emitted lightdetected by each of the plurality of cameras lies within the range ofvalues for a given pixel, the electronic circuitry sends a signal toremove the corresponding cell from the given microchannel. In somecases, the circuitry is implemented as firmware. In some cases, thecircuitry is implemented as a field programmable gate array (FPGA).

In some instances, the range of values for each detector may be unknownat the outset of the scanning procedure. For instance, the range ofvalues may depend on the cell type (e.g. red blood cell, white bloodcell, etc). The range of values may depend on operating parameters ofthe system (e.g. age-related degradation of optical components). Thus,it may be beneficial to determine acceptable ranges of values uponreceiving a new cassette in the system. For instance, such ranges may bedetermined by scanning the plurality of excitation light sources over asample population of cells and determining the intensities at each pixelof each camera for the population of cells. The population of cells maycomprise more than 1,000, more than 10,000, more than 100,000, or morethan 1,000,000 cells. The population of cells may lie in a range definedby any two of the preceding values. The range of values may be chosensuch that only a subpopulation of the cells from the sample populationgives rise to fluorescence intensities falling within the range ofvalues. For instance, the range of values for each pixel of each cameramay be chosen such that less than 1%, less than 5%, less than 10%, lessthan 20%, or less than 50% of the cells give rise to fluorescenceintensities at each pixel at each camera that fall within the range ofvalues. The range of values may be chosen to lie within a range definedby any two of the preceding values. These ranges of values may then beused to determine whether a given cell should be removed from a givenmicrochannel during a scan of the entire cassette.

When the electronic circuitry makes a determination that a given cell ina given microchannel meets the criteria for removal from themicrochannel, a signal may be sent to direct light from an extractionlaser source 1430 to the microchannel. In some cases, the extractionlaser and the circuitry may be synchronized to a shared clock. Theextraction laser source may emit light at a wavelength that allowsextraction of a cell from a microchannel. For instance, the extractionlaser source may emit light at a wavelength that is absorbed by aparticle in a microchannel, as described herein. The extraction lasersource may emit light at a wavelength in the ultraviolet, visible, ornear infrared region of the electromagnetic spectrum. The extractionlaser source may emit light in a range of wavelengths from about 200 nmto about 2000 nm. The extraction laser source may comprise a continuouswave laser source. The extraction source may comprise a quasi-continuouswave laser source. The extraction laser source may comprise a pulsedlaser source. The pulsed laser source may emit laser pulses having aduration shorter than 10 fs, shorter than 100 fs, shorter than 1 μs,shorter than 10 μs, shorter than 100 μs, shorter than 1 ns, shorter than10 ns, shorter than 100 ns, shorter than 1 μs, shorter than 10 μs,shorter than 100 μs, or shorter than 1 ms. The pulsed laser source mayemit laser pulses having a duration lying in a range defined by any twoof the preceding values. In some cases, the pulsed laser source may emitlaser pulses having a duration within the range from about 1 fs to about100 μs. In some cases, the pulsed laser source may emit laser pulseshaving a duration within the range from about 100 ns to about 10 μs. Thepulsed laser source may emit laser pulses having an adjustable duration.

The pulsed laser source may emit laser pulses with a repetition rate ofless than 1 kHz, less than 10 kHz, less than 100 kHz, less than 1 MHz,less than 10 MHz, less than 100 MHz, or less than 1 GHz. The pulsedlaser source may emit laser pulses with a repetition rate lying in arange defined by any two of the preceding values. In some cases, thepulsed laser source may emit pulses with a repetition rate within therange from about 10 KHz to about 1 MHz. In some cases, each pulse mayproduce less than 1 nJ, less than 10 nJ, less than 100 nJ, less than 1μJ, less than 10 μJ, less than 100 μJ, less than 1 mJ, or less than 10mJ of energy. Each pulse may produce an energy lying in a range definedby any two of the preceding values. In some cases, each pulse mayproduce an energy lying in the range from about 100 nJ to about 1 mJ.The pulsed laser source may emit a peak power in the range from 0.1 W to10⁷ W.

The pulsed laser source may be a fiber laser source. The pulsed lasersource may be a pulse-on-demand fiber laser source. The pulsed lasersource may be a master oscillator fiber amplifier (MOPA) laser source.The pulsed laser source may be a doped fiber laser source. The pulsedlaser source may be a rare earth ion doped fiber laser source. Thepulsed laser source may be a ytterbium doped fiber laser source. Thepulsed laser source may be a laser utilizing a doped crystal gainmedium. The pulsed laser source may be a laser utilizing aneodymium-doped crystal gain medium. The pulsed laser source may be aNd:YAG laser. The pulsed laser source may be a Nd:YVO₄ laser. The pulsedlaser source may be a semiconductor laser. The pulsed laser source maybe a diode laser. The pulsed laser source may be a vertical cavitysurface emitting laser (VCSEL). The pulsed laser source may be a VCSELarray. The pulsed laser source may be a gas laser. The pulsed lasersource may be a CO₂ laser. The pulsed laser source may be an excimerlaser. The pulsed laser source may employ Q-switching. The pulsed lasersource may employ mode locking. The pulsed laser source may be anypulsed laser source as is known to one having skill in the art.

The pulsed laser source may direct a laser pulse to a microchannel inresponse to a signal from electronic circuitry in response tointensities of light detector at each of the plurality of detectors. Thepulse may be directed to the microchannel using an acousto-opticalmodulator (AOM), electro-optic modular (EOM), or any modulation deviceas may be known to one having skill in the art. For instance, themodulation device may be configured to pass the zero-order diffractedbeam only in response to a signal that the extraction laser shouldremove a cell from a microchannel; at all other times, the modulationdevice may be configured to pass the first-order diffracted beam orhigher-order diffracted beam. In some cases, the pulsed laser mayproduce pulses only in response to a signal that the extraction lasershould remove a cell from a microchannel. For instance, a doped fiberlaser may be operated in a reverse-biased configuration until receivinga signal to direct a pulse to the microchannel; upon receiving thesignal to direct a pulse to the microchannel, the doped fiber laser maybe operated in the forward-biased configuration.

The extraction laser may be directed to a galvonometer scanner block1440. The galvonometer scanner block may be configured to rapidly scanlight from the extraction laser along a curved focal plane perpendicularto its axes of rotation. The galvonometer may be scanned until receivinga signal from the circuitry to fire a laser pulse at a desired firingposition. The galvonometer scanner block may be configured to wait for asignal from the circuitry before directing the extraction laser to adesired firing position. Following direction of the extraction beam bythe galvonometer scanner block, F-theta relays lenses 1450 and 1452produce a focal plane that is substantially flat. After reflection fromthe galvonometer scanner block and refraction through the F-thetalenses, light from the extraction laser is directed to the cassette 100.The F-theta lenses may be configured to focus the plurality ofextraction beams to a microchannel in the cassette. When the extractionlaser is directed to a microchannel, energy from the laser causescavitation of the liquid sample in which a cell is suspended. Thiscauses the removal of the cell from the microchannel.

The system may be configured to scan the extraction beam. In some cases,the system may be configured to scan the microchannels at a rate of morethan 10,000 channels per second, more than 50,000 channels per second,more than 100,000 channels per second, more than 500,000 channels persecond, more than 1,000,000 channels per second, more than 5,000,000channels per second, more than 10,000,000 channels per second, more than50,000,000 channels per second, or more than 100,000,000 channels persecond. The system may be configured to scan the microchannels at a ratelying in a range defined by any two of the preceding values. In somecases, the system may be configured to scan the microchannels at a ratewithin a range from about 3,000 to about 300,000,000 channels persecond.

System 1400 may scan the substrate at a rate greater than 1,000,000microchannels per second, greater than 2,000,000 microchannels persecond, or greater than 3,000,000 microchannels per second. System 1400may scan the substrate at a rate that is within a range defined by anytwo of the preceding values. System 1400 may extract target particlesfrom the substrate at a rate greater than 500,000 microchannels persecond, greater than 600,000 microchannels per second, greater than700,000 microchannels per second, greater than 800,000 microchannels persecond, greater than 900,000 microchannels per second, or greater than1,000,000 microchannels per second. System 1400 may extract targetparticles at a rate that is within a range defined by any two of thepreceding values. System 1100 may extract the target particles such thata collection of extracted target particles has a purity greater than90%, greater than 95%, or greater than 99%. System 1400 may extract thetarget particles such that the collection of extracted target particleshas a purity that is within a range defined by any two of the precedingvalues.

Though shown as forming a single device in FIG. 14, system 1400 may beconfigured such that the fluorescence subsystem (comprising elements1410, 1420, 1422, 1424, 1460, 1470, 1480, 1482, 1484, and 1490) arearranged as a fluorescence device and the extraction subsystem(comprising elements 1430, 1440, 1450, 1452, 1460, and 1470) arearranged as an extraction device. In such an arrangement, the substrate100 may be subjected to a fluorescence analysis as described hereinusing the fluorescence device. Following the fluorescence analysis, thesubstrate may be transferred to the extraction device for extraction oflocations of interest on the substrate identified during thefluorescence analysis. Proper alignment of the substrate in each of thefluorescence device and the extraction device may be achieved byreferring to one or more fiducial markers on the substrate. In somecases, arranging system 1400 on two devices may require duplication ofone or more elements of system 1400. For instance, elements 1460 and1470 may be required on both the fluorescence device and the extractiondevice.

Alternative Fluorescence Detection System

FIG. 17 shows a schematic for an alternative fluorescence detectionsystem 1700. The system 1700 may be used in place of any otherfluorescence detection system described herein. For instance, the system1700 may be used in place of any subset of the set of components 1110,1112, 1120, 1122, 1124, 1132, 1150, 1160, 1162, 1164, 1170, and 1172 ofFIG. 11, any subset of the set of components 1110, 1112, 1120, 1122,1124, 1140, 1150, 1160, 1162, 1174, 1170, and 1172 of FIG. 12, anysubset of the set of components 1110, 1112, 1120, 1122, 1124, 1140,1150, 1164, 1166, 1168, 1170, and 1172 of FIG. 13, or any subset of theset of components 1410, 1420, 1422, 1424, 1460, 1470, 1480, 1482, 1484,and 1490 of FIG. 14.

In contrast to certain epifluorescence microscope systems, the system1700 may operate by sending excitation light along an optical path thatdoes not pass through an objective lens and by collecting emittedfluorescence light through the objective lens. The system 1700 maycomprise an excitation light source 1710. The excitation light sourcemay be any excitation light source described herein. For instance, theexcitation light source may be any excitation laser source describedherein. The excitation light source may produce light at any wavelengthdescribed herein.

The system 1700 may comprise one or more mirrors for directing lightfrom the excitation light source to a beam expander 1730. For instance,the system may comprise mirrors 1720 a, 1720 b, and 1720 c. Though shownas comprising three mirrors in FIG. 17, the system may comprise anynumber of mirrors for directing light to the beam expander, such as 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mirrors. The mirrors maycomprise one or more coatings for enhancing reflectivity, such as one ormore dielectric coatings as are known to one having skill in the art.

The beam expander 1730 may expand the beam waist of the excitation lightby a factor of 1, 2, 5, or 10. The beam expander may expand the beamwaist of the excitation light by a factor that is within a range definedby any two of the preceding values.

The beam expander may direct expanded excitation light to a scanningmechanism 1740. The scanning mechanism may be similar to any scanningmechanism described herein. For instance, the scanning mechanism maycomprise a polygon mirror as described herein. The scanning mechanismmay comprise a galvanometer as described herein. The scanning mechanismmay direct the excitation light to one or more locations on a substrate100. The substrate may be any substrate described herein (such as one ormore microchannels on a microchannel array described herein).

The scanning mechanism may direct the excitation light to the one ormore locations on the substrate such that the excitation light does notpass through an objective lens 1750. The scanning mechanism may directthe excitation light to the one or more locations on the substrate suchthat the excitation light hits the one or more locations on thesubstrate at an angle to the normal. Configuring the system in thismanner may reduce noise associated with the fluorescence system. Forinstance, such a configuration may reduce speckle noise or noiseassociated with auto-fluorescence of the objective lens. In someembodiments, such a configuration reduces background fluorescence by 20%or more. The configuration may reduce background fluorescence by 30% ormore, 40% or more, 50% or more, 60% or more, or 70% or more. In anexemplary embodiment, such a configuration reduces backgroundfluorescence by 60% or more. Configuring the system in this matter mayreduce speckle noise detected by the light detector by 30% or more, 40%or more, 50%, or more, 60% or more, or 70% or more. In an exemplaryembodiment, the configuration reduces speckle noise detected by thelight detector by 50% or more.

The excitation light may interact with the locations on the substrate toproduce fluorescence light, as described herein.

The objective lens 1750 may collect the fluorescence light. Theobjective lens may comprise any objective lens described herein. Theobjective lens may direct fluorescence light to one or morebeamsplitters 1760 a, 1760 b, and 1760 c, one or more filters 1770 a,1770 b, and 1770 c, one or more lenses (such as one or more tube lenses)1780 a, 1780 b, 1780 c, and one or more light detectors (such asphotodiodes, CCD cameras, or CMOS cameras) 1790 a, 1790 b, and 1790 c.Though shown as comprising three beamsplitters, three filters, threelenses, and three light detectors in FIG. 17, the fluorescence detectionsystem 1700 may comprise any number of beamsplitters, such as 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10 beamsplitters, any number offilters, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 filters,any number of lenses, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10 lenses, and any number of light detectors, such as 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10 light detectors. The beamsplittersmay comprise any beamsplitters described herein. The filters maycomprise any filters described herein. The lenses may comprise anylenses described herein. The light detectors may comprise any lightdetectors described herein.

Digital Processing Device

In some embodiments, the platforms, systems, media, and methodsdescribed herein include a digital processing device, or use of thesame. In further embodiments, the digital processing device includes oneor more hardware central processing units (CPUs), general purposegraphics processing units (GPGPUs), or field programmable gate arrays(FPGAs) that carry out the device's functions. In still furtherembodiments, the digital processing device further comprises anoperating system configured to perform executable instructions. In someembodiments, the digital processing device is optionally connected acomputer network. In further embodiments, the digital processing deviceis optionally connected to the Internet such that it accesses the WorldWide Web. In still further embodiments, the digital processing device isoptionally connected to a cloud computing infrastructure. In otherembodiments, the digital processing device is optionally connected to anintranet. In other embodiments, the digital processing device isoptionally connected to a data storage device.

In accordance with the description herein, suitable digital processingdevices include, by way of non-limiting examples, server computers,desktop computers, laptop computers, notebook computers, sub-notebookcomputers, netbook computers, netpad computers, set-top computers, mediastreaming devices, handheld computers, Internet appliances, mobilesmartphones, tablet computers, personal digital assistants, video gameconsoles, and vehicles. Those of skill in the art will recognize thatmany smartphones are suitable for use in the system described herein.Those of skill in the art will also recognize that select televisions,video players, and digital music players with optional computer networkconnectivity are suitable for use in the system described herein.Suitable tablet computers include those with booklet, slate, andconvertible configurations, known to those of skill in the art.

In some embodiments, the digital processing device includes an operatingsystem configured to perform executable instructions. The operatingsystem is, for example, software, including programs and data, whichmanages the device's hardware and provides services for execution ofapplications. Those of skill in the art will recognize that suitableserver operating systems include, by way of non-limiting examples,FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle®Solaris®, Windows Server®, and Novell® NetWare®. Those of skill in theart will recognize that suitable personal computer operating systemsinclude, by way of non-limiting examples, Microsoft® Windows®, Apple®Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU/Linux®. Insome embodiments, the operating system is provided by cloud computing.Those of skill in the art will also recognize that suitable mobile smartphone operating systems include, by way of non-limiting examples, Nokia®Symbian® OS, Apple® iOS®, Research In Motion® BlackBerry OS®, Google®Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS,Linux®, and Palm® WebOS®. Those of skill in the art will also recognizethat suitable media streaming device operating systems include, by wayof non-limiting examples, Apple TV®, Roku®, Boxee®, Google TV®, GoogleChromecast®, Amazon Fire®, and Samsung® HomeSync®. Those of skill in theart will also recognize that suitable video game console operatingsystems include, by way of non-limiting examples, Sony® PS3®, Sony PS4®,Microsoft® Xbox 360®, Microsoft Xbox One, Nintendo® Wii®, Nintendo® WiiU®, and Ouya®.

In some embodiments, the device includes a storage and/or memory device.The storage and/or memory device is one or more physical apparatusesused to store data or programs on a temporary or permanent basis. Insome embodiments, the device is volatile memory and requires power tomaintain stored information. In some embodiments, the device isnon-volatile memory and retains stored information when the digitalprocessing device is not powered. In further embodiments, thenon-volatile memory comprises flash memory. In some embodiments, thenon-volatile memory comprises dynamic random-access memory (DRAM). Insome embodiments, the non-volatile memory comprises ferroelectric randomaccess memory (FRAM). In some embodiments, the non-volatile memorycomprises phase-change random access memory (PRAM). In otherembodiments, the device is a storage device including, by way ofnon-limiting examples, CD-ROMs, DVDs, flash memory devices, magneticdisk drives, magnetic tapes drives, optical disk drives, and cloudcomputing based storage. In further embodiments, the storage and/ormemory device is a combination of devices such as those disclosedherein.

In some embodiments, the digital processing device includes a display tosend visual information to a user. In some embodiments, the display is acathode ray tube (CRT). In some embodiments, the display is a liquidcrystal display (LCD). In further embodiments, the display is a thinfilm transistor liquid crystal display (TFT-LCD). In some embodiments,the display is an organic light emitting diode (OLED) display. Invarious further embodiments, on OLED display is a passive-matrix OLED(PMOLED) or active-matrix OLED (AMOLED) display. In some embodiments,the display is a plasma display. In other embodiments, the display is avideo projector. In still further embodiments, the display is acombination of devices such as those disclosed herein.

In some embodiments, the digital processing device includes an inputdevice to receive information from a user. In some embodiments, theinput device is a keyboard. In some embodiments, the input device is apointing device including, by way of non-limiting examples, a mouse,trackball, track pad, joystick, game controller, or stylus. In someembodiments, the input device is a touch screen or a multi-touch screen.In other embodiments, the input device is a microphone to capture voiceor other sound input. In other embodiments, the input device is a videocamera or other sensor to capture motion or visual input. In furtherembodiments, the input device is a Kinect, Leap Motion, or the like. Instill further embodiments, the input device is a combination of devicessuch as those disclosed herein.

Referring to FIG. 16, in a particular embodiment, an exemplary digitalprocessing device 1601 is programmed or otherwise configured to operatea laser scanning cell sorting device. The device 1601 can regulatevarious aspects of the laser scanning cell sorting of the presentdisclosure, such as, for example, performing processing steps. In thisembodiment, the digital processing device 1601 includes a centralprocessing unit (CPU, also “processor” and “computer processor” herein)1605, which can be a single core or multi core processor, or a pluralityof processors for parallel processing. The digital processing device1601 also includes memory or memory location 1610 (e.g., random-accessmemory, read-only memory, flash memory), electronic storage unit 1615(e.g., hard disk), communication interface 1620 (e.g., network adapter)for communicating with one or more other systems, and peripheral devices1625, such as cache, other memory, data storage and/or electronicdisplay adapters. The memory 1610, storage unit 1615, interface 1620 andperipheral devices 1625 are in communication with the CPU 1605 through acommunication bus (solid lines), such as a motherboard. The storage unit1615 can be a data storage unit (or data repository) for storing data.The digital processing device 1601 can be operatively coupled to acomputer network (“network”) 1630 with the aid of the communicationinterface 1620. The network 1630 can be the Internet, an internet and/orextranet, or an intranet and/or extranet that is in communication withthe Internet. The network 1630 in some cases is a telecommunicationand/or data network. The network 1630 can include one or more computerservers, which can enable distributed computing, such as cloudcomputing. The network 1630, in some cases with the aid of the device1601, can implement a peer-to-peer network, which may enable devicescoupled to the device 1601 to behave as a client or a server.

Continuing to refer to FIG. 16, the CPU 1605 can execute a sequence ofmachine-readable instructions, which can be embodied in a program orsoftware. The instructions may be stored in a memory location, such asthe memory 1610. The instructions can be directed to the CPU 1605, whichcan subsequently program or otherwise configure the CPU 1605 toimplement methods of the present disclosure. Examples of operationsperformed by the CPU 1605 can include fetch, decode, execute, and writeback. The CPU 1605 can be part of a circuit, such as an integratedcircuit. One or more other components of the device 1601 can be includedin the circuit. In some cases, the circuit is an application specificintegrated circuit (ASIC) or a field programmable gate array (FPGA).

Continuing to refer to FIG. 16, the storage unit 1615 can store files,such as drivers, libraries and saved programs. The storage unit 1615 canstore user data, e.g., user preferences and user programs. The digitalprocessing device 1601 in some cases can include one or more additionaldata storage units that are external, such as located on a remote serverthat is in communication through an intranet or the Internet.

Continuing to refer to FIG. 16, the digital processing device 1601 cancommunicate with one or more remote computer systems through the network1630. For instance, the device 1601 can communicate with a remotecomputer system of a user. Examples of remote computer systems includepersonal computers (e.g., portable PC), slate or tablet PCs (e.g.,Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g.,Apple® iPhone, Android-enabled device, Blackberry®), or personal digitalassistants.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the digital processing device 1601, such as, for example, onthe memory 1610 or electronic storage unit 1615. The machine executableor machine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1605. In some cases, thecode can be retrieved from the storage unit 1615 and stored on thememory 1610 for ready access by the processor 1605. In some situations,the electronic storage unit 1315 can be precluded, andmachine-executable instructions are stored on memory 1610.

Non-Transitory Computer Readable Storage Medium

In some embodiments, the platforms, systems, media, and methodsdisclosed herein include one or more non-transitory computer readablestorage media encoded with a program including instructions executableby the operating system of an optionally networked digital processingdevice. In further embodiments, a computer readable storage medium is atangible component of a digital processing device. In still furtherembodiments, a computer readable storage medium is optionally removablefrom a digital processing device. In some embodiments, a computerreadable storage medium includes, by way of non-limiting examples,CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic diskdrives, magnetic tape drives, optical disk drives, cloud computingsystems and services, and the like. In some cases, the program andinstructions are permanently, substantially permanently,semi-permanently, or non-transitorily encoded on the media.

Computer Program

In some embodiments, the platforms, systems, media, and methodsdisclosed herein include at least one computer program, or use of thesame. A computer program includes a sequence of instructions, executablein the digital processing device's CPU, written to perform a specifiedtask. Computer readable instructions may be implemented as programmodules, such as functions, objects, Application Programming Interfaces(APIs), data structures, and the like, that perform particular tasks orimplement particular abstract data types. In light of the disclosureprovided herein, those of skill in the art will recognize that acomputer program may be written in various versions of variouslanguages.

The functionality of the computer readable instructions may be combinedor distributed as desired in various environments. In some embodiments,a computer program comprises one sequence of instructions. In someembodiments, a computer program comprises a plurality of sequences ofinstructions. In some embodiments, a computer program is provided fromone location. In other embodiments, a computer program is provided froma plurality of locations. In various embodiments, a computer programincludes one or more software modules. In various embodiments, acomputer program includes, in part or in whole, one or more webapplications, one or more mobile applications, one or more standaloneapplications, one or more web browser plug-ins, extensions, add-ins, oradd-ons, or combinations thereof.

Web Application

In some embodiments, a computer program includes a web application. Inlight of the disclosure provided herein, those of skill in the art willrecognize that a web application, in various embodiments, utilizes oneor more software frameworks and one or more database systems. In someembodiments, a web application is created upon a software framework suchas Microsoft®.NET or Ruby on Rails (RoR). In some embodiments, a webapplication utilizes one or more database systems including, by way ofnon-limiting examples, relational, non-relational, object oriented,associative, and XML database systems. In further embodiments, suitablerelational database systems include, by way of non-limiting examples,Microsoft® SQL Server, mySQL™, and Oracle®. Those of skill in the artwill also recognize that a web application, in various embodiments, iswritten in one or more versions of one or more languages. A webapplication may be written in one or more markup languages, presentationdefinition languages, client-side scripting languages, server-sidecoding languages, database query languages, or combinations thereof. Insome embodiments, a web application is written to some extent in amarkup language such as Hypertext Markup Language (HTML), ExtensibleHypertext Markup Language (XHTML), or eXtensible Markup Language (XML).In some embodiments, a web application is written to some extent in apresentation definition language such as Cascading Style Sheets (CSS).In some embodiments, a web application is written to some extent in aclient-side scripting language such as Asynchronous Javascript and XML(AJAX), Flash® Actionscript, Javascript, or Silverlight®. In someembodiments, a web application is written to some extent in aserver-side coding language such as Active Server Pages (ASP),ColdFusion®, Perl, Java™, JavaServer Pages (JSP), Hypertext Preprocessor(PHP), Python™, Ruby, Tcl, Smalltalk, WebDNA®, or Groovy. In someembodiments, a web application is written to some extent in a databasequery language such as Structured Query Language (SQL). In someembodiments, a web application integrates enterprise server productssuch as IBM® Lotus Domino®. In some embodiments, a web applicationincludes a media player element. In various further embodiments, a mediaplayer element utilizes one or more of many suitable multimediatechnologies including, by way of non-limiting examples, Adobe® Flash®,HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, Java™, and Unity®.

Mobile Application

In some embodiments, a computer program includes a mobile applicationprovided to a mobile digital processing device. In some embodiments, themobile application is provided to a mobile digital processing device atthe time it is manufactured. In other embodiments, the mobileapplication is provided to a mobile digital processing device via thecomputer network described herein.

In view of the disclosure provided herein, a mobile application iscreated by techniques known to those of skill in the art using hardware,languages, and development environments known to the art. Those of skillin the art will recognize that mobile applications are written inseveral languages. Suitable programming languages include, by way ofnon-limiting examples, C, C++, C#, Objective-C, Java™ Javascript,Pascal, Object Pascal, Python™, Ruby, VB.NET, WML, and XHTML/HTML withor without CSS, or combinations thereof.

Suitable mobile application development environments are available fromseveral sources. Commercially available development environmentsinclude, by way of non-limiting examples, AirplaySDK, alcheMo,Appcelerator®, Celsius, Bedrock, Flash Lite, .NET Compact Framework,Rhomobile, and WorkLight Mobile Platform. Other development environmentsare available without cost including, by way of non-limiting examples,Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile devicemanufacturers distribute software developer kits including, by way ofnon-limiting examples, iPhone and iPad (iOS) SDK, Android™ SDK,BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, andWindows® Mobile SDK.

Those of skill in the art will recognize that several commercial forumsare available for distribution of mobile applications including, by wayof non-limiting examples, Apple® App Store, Google® Play, ChromeWebStore, BlackBerry® App World, App Store for Palm devices, App Catalogfor webOS, Windows® Marketplace for Mobile, Ovi Store for Nokia®devices, Samsung® Apps, and Nintendo® DSi Shop.

Standalone Application

In some embodiments, a computer program includes a standaloneapplication, which is a program that is run as an independent computerprocess, not an add-on to an existing process, e.g., not a plug-in.Those of skill in the art will recognize that standalone applicationsare often compiled. A compiler is a computer program(s) that transformssource code written in a programming language into binary object codesuch as assembly language or machine code. Suitable compiled programminglanguages include, by way of non-limiting examples, C, C++, Objective-C,COBOL, Delphi, Eiffel, Java™, Lisp, Python™, Visual Basic, and VB.NET,or combinations thereof. Compilation is often performed, at least inpart, to create an executable program. In some embodiments, a computerprogram includes one or more executable complied applications.

Web Browser Plug-in

In some embodiments, the computer program includes a web browser plug-in(e.g., extension, etc.). In computing, a plug-in is one or more softwarecomponents that add specific functionality to a larger softwareapplication. Makers of software applications support plug-ins to enablethird-party developers to create abilities which extend an application,to support easily adding new features, and to reduce the size of anapplication. When supported, plug-ins enable customizing thefunctionality of a software application. For example, plug-ins arecommonly used in web browsers to play video, generate interactivity,scan for viruses, and display particular file types. Those of skill inthe art will be familiar with several web browser plug-ins including,Adobe® Flash® Player, Microsoft® Silverlight®, and Apple® QuickTime®. Insome embodiments, the toolbar comprises one or more web browserextensions, add-ins, or add-ons. In some embodiments, the toolbarcomprises one or more explorer bars, tool bands, or desk bands.

In view of the disclosure provided herein, those of skill in the artwill recognize that several plug-in frameworks are available that enabledevelopment of plug-ins in various programming languages, including, byway of non-limiting examples, C++, Delphi, Java™, PHP, Python™, and VB.NET, or combinations thereof.

Web browsers (also called Internet browsers) are software applications,designed for use with network-connected digital processing devices, forretrieving, presenting, and traversing information resources on theWorld Wide Web. Suitable web browsers include, by way of non-limitingexamples, Microsoft® Internet Explorer®, Mozilla® Firefox®, Google®Chrome, Apple® Safari®, Opera Software® Opera®, and KDE Konqueror. Insome embodiments, the web browser is a mobile web browser. Mobile webbrowsers (also called microbrowsers, mini-browsers, and wirelessbrowsers) are designed for use on mobile digital processing devicesincluding, by way of non-limiting examples, handheld computers, tabletcomputers, netbook computers, subnotebook computers, smartphones, musicplayers, personal digital assistants (PDAs), and handheld video gamesystems. Suitable mobile web browsers include, by way of non-limitingexamples, Google® Android® browser, RIM BlackBerry® Browser, Apple®Safari®, Palm® Blazer, Palm® WebOS® Browser, Mozilla® Firefox® formobile, Microsoft® Internet Explorer® Mobile, Amazon® Kindle® Basic Web,Nokia® Browser, Opera Software® Opera® Mobile, and Sony® PSP™ browser.

Software Modules

In some embodiments, the platforms, systems, media, and methodsdisclosed herein include software, server, and/or database modules, oruse of the same. In view of the disclosure provided herein, softwaremodules are created by techniques known to those of skill in the artusing machines, software, and languages known to the art. The softwaremodules disclosed herein are implemented in a multitude of ways. Invarious embodiments, a software module comprises a file, a section ofcode, a programming object, a programming structure, or combinationsthereof. In further various embodiments, a software module comprises aplurality of files, a plurality of sections of code, a plurality ofprogramming objects, a plurality of programming structures, orcombinations thereof. In various embodiments, the one or more softwaremodules comprise, by way of non-limiting examples, a web application, amobile application, and a standalone application. In some embodiments,software modules are in one computer program or application. In otherembodiments, software modules are in more than one computer program orapplication. In some embodiments, software modules are hosted on onemachine. In other embodiments, software modules are hosted on more thanone machine. In further embodiments, software modules are hosted oncloud computing platforms. In some embodiments, software modules arehosted on one or more machines in one location. In other embodiments,software modules are hosted on one or more machines in more than onelocation.

Databases

In some embodiments, the platforms, systems, media, and methodsdisclosed herein include one or more databases, or use of the same. Inview of the disclosure provided herein, those of skill in the art willrecognize that many databases are suitable for storage and retrieval ofinformation. In various embodiments, suitable databases include, by wayof non-limiting examples, relational databases, non-relationaldatabases, object oriented databases, object databases,entity-relationship model databases, associative databases, and XMLdatabases. Further non-limiting examples include SQL, PostgreSQL, MySQL,Oracle, DB2, and Sybase. In some embodiments, a database isinternet-based. In further embodiments, a database is web-based. Instill further embodiments, a database is cloud computing-based. In otherembodiments, a database is based on one or more local computer storagedevices.

EXAMPLES Example 1: Macro-Gel Isolation

As presented in FIG. 6, opaque particles and cells may be separated in amicrochannel of a substrate in order to protect the cells fromelectromagnetic radiation during extraction. First, approximately 1 g ofagarose (Sigma-Aldrich, Cat No: A9539) was added to approximately 100 mLof distilled water. The mixture was heated in a microwave oven and themixture was stirred intermittently to dissolve the agarose. Opaque beads(Dynabeads M-270 Epoxy, Thermo Fisher, Cat No. 14301) were added to themixture and the beads were mixed to make a uniform distribution. Themixture was applied onto the surface of a micropore array and themixture was allowed to enter into the pores by surface tension. Themixture in the pores was allowed to cool for at least approximately 20min at room temperature and to set inside the pores to make a gelinfused micropore array. The gel infused micropore array may be storedin approximately 4° C. for future use. Then, a reservoir on one side ofthe gel infused micropore array was created and buffer QG (QIAGEN, CatNo; 19063) was added. Buffer QG was allowed to sit and dissolve aportion of the agarose on one side of the pore for approximately 5 min.The QG buffer was washed off with PBS (Thermo Fisher, Cat No: 10010023)and with 2% BSA (VWR, Cat No. 97063-626), and this step was repeatedtwice. A cell suspension solution was added to the side of the pore thatwas dissolved (“etched out”). The cell suspension solution was allowedto sit for approximately 10 min and allowed the cells to sediment intothe pores. The micropore array was washed twice with PBS and 2% BSAsolution to get rid of extra cells stuck to the surface of the pore. Thepore was inverted and allowed to sit for approximately 10 min. The cellssediment on the meniscus due to gravity.

Example 2: Micro-Gel Isolation

As presented in FIG. 7B, opaque particles and cells may be separated ina microchannel of a substrate in order to protect the cells fromelectromagnetic radiation during extraction. First, cells in PBS and 2%BSA were added to beads that have a magnetic core and an agarose shell(Cube-BioTech) in a volume of approximately 20 μL of 50% (V/V) per 50 μLof cell suspension. The suspension was loaded onto a micropore array andallowed the suspension to enter into the pores by capillary action. Astrong neodymium magnet (cylindrical shape diameter 6.5 mm, length 25mm, strength N52) was applied for approximately 5 min to the top of thearray in order to cause the magnetic beads to settle on the uppermeniscus. Then, the magnet was removed. The cells and beads were allowedto settle for approximately 10 min, and allowed a distinct layer ofcells to settle on the lower meniscus and a layer of beads to settle onthe top.

Example 3: In-Pore Spacer

As presented in FIG. 8A, magnetic particles and cells may be separatedin a microchannel of a substrate in order to protect the cells fromelectromagnetic radiation during extraction. First, cells in PBS and 2%BSA were added to approximately 10 μL of 0.25×10⁹ beads/mL in 100 μL oftransparent silica (Bangs Laboratories, Inc., Cat No: SS05N, size 3.48μm) and approximately 2 μL of 2×10⁹ beads/mL in 100 μL of opaquemagnetic beads (Dynabeads® M-270 Epoxy, Thermo Fisher, CAT No: 14301).The suspension was loaded onto a micropore array and allowed thesuspension to enter into the pores by capillary action. A strongneodymium magnet (cylindrical shape diameter 6.5 mm, length 25 mm,strength N52) was applied for approximately 5 min to the top of thearray in order to cause the magnetic beads to settle on the uppermeniscus. Then, the magnet was removed. The cells and beads were allowedto settle for approximately 10 min, and allowed a distinct layer ofcells and transparent silica beads to settle on the lower meniscus and alayer of beads to settle on the top.

Example 4: Sequential Loading

As presented in FIG. 9A, cells may be loaded into an array sequentiallywith opaque beads or loaded in a mixed batch in order to quantify cells.First, load the micropore array with cells suspended in PBS and 2% FBS.Allow the cells to settle for 10 min. Then, add beads to the microporearray with a concentration between 1 to 2 billion per mL. Allow thecontents of the microarray to sit for at least 10 min to allow the beadsto settle into the pores and rest on top of the cells.

Example 5: Hanging Bead

Put 60 μL of cell suspension on the bottom of a clean glass slide, sothat the droplet is inverted. The droplet stays in place due to thehydrophilicity and the surface tension balancing out the gravitationalpull. Allow the droplet to settle for 10 min. Apply the micropore arrayto the hanging drop. The micropore array may be applied with aZ-translation stage or by hand. The first end, middle portion, andsecond end of the micropore array should be substantially in the sameplane and flat when applied to the hanging drop. Allow the cellsuspension to enter the micropore array by capillary action. Put a PDMSreservoir on the side that touched the hanging drop. Fill up thereservoir with 100 μL of buffer for hydration. Settle for 10 min beforeimaging.

Example 6: Flooding

The PDMS reservoir (diameter 7 mm and height 3 mm) was fixed to the topof a micropore glass array. Approximately 60 μm of cell suspension (ofdifferent concentration) was added to the top of the micropore glassarray. Approximately 10 μL of cell suspension was taken out from the topof a micropore glass array. The PDMS reservoir was filled withapproximately 100 μL of buffer to keep the pores hydrated. Settled forapproximately 10 min before imaging.

Example 7: Sonication

Approximately 100 μL of buffer (PBS+0.2% BSA) was added to a 20 μm glassmicrochip. Remove approximately 20 μL of buffer from the PDMS reservoir.Approximately 15 μL of re-suspended beads was added at a knownconcentration. Waited for approximately 10 min. Imaged with microscopy.The Tide sonicator (sonicator frequency 40 KHz) was applied on the sideof the chip. Waited for approximately 10 min. Imaged with microscopy. Astrong neodymium magnet was applied at the bottom of the chip to pullthe beads to the bottom. Imaged with microscopy.

Example 8: Fluorescence Displacement

A micropore array was loaded with cells and COMPEL™ beads (5 μL of0.6×10⁹ in 100 μL of cell suspension, Bangs Laboratories, Inc., Cat No:UMC3F, size 2.85 μm, fluorescent emission: green) suspended in PBS and2% FBS. A strong neodymium magnet (cylindrical shape diameter 6.5 mm,length 25 mm, strength N52) was added at top of the array forapproximately 5 min to cause the magnetic beads to settle on the uppermeniscus. The magnet was removed and allowed the cells and beads tosettle for approximately 10 min. Imaged the cells settled on the lowermeniscus.

Example 9: Extraction

FIG. 15 shows the optimal pulse power settings for laser extraction ofcells from a microchannel array. A study was conducted to determineextraction laser settings that would allow the expulsion of fluid fromdesired wells within a microchannel array. The study was conducted usinga IPG Photonics YLPN-1-1×120-100-M adjustable pulse duration nanosecondytterbium-doped fiber laser. The laser was focused to a beam diameter of6.55 μm and scanned across a microchannel array using a SCANLabhurrySCAN II 14 galvonometer scanner. The extraction light was focusedonto a flat plane using a Linos F-Theta-Ronar lens with a 163 mm focallength. Direction of the extraction laser pulses was controlled using aLanmark Maestro 3000 LEC-1 Ethernet smart controller with a built-in IPGPhotonics laser extension board. The controller was programmed using theLanmark WinLase LAN v 5.1.9.17 software. The pulse duration wasarbitrarily set to 4 ns. The pulse repetition rate was set at 100 kHz.

Trials were performed with varying levels of average laser power. Asshown in the left pane of FIG. 15, an average laser power of 2.0 Wresulted in only marginal expulsion of fluid from desired microchannels(light spots in the array of microchannels). As shown in the right pane,an average laser power of 3.3 W resulted in the complete expulsion offluid from the desired microchannels. As shown in the central pane, theoptimal average laser power was determined to be 2.3 W, corresponding toan energy of 23 μJ for each laser pulse.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of sorting cells, comprising screeningcellular material to identify cells with a desired phenotype wherein thecellular material is screened at a rate of 1,000,000 cells per second orgreater.
 2. A method of sorting cells, comprising screening cellularmaterial to identify cells with a desired phenotype, wherein thecellular material is screened at a rate of 50,000 cells per second orgreater; and extracting said cells of a desired phenotype from saidcellular material at a rate of 100,000 cells per second or greater. 3.The method of claim 2, wherein said cells of a desired phenotype areextracted at a rate of 300,000 cells per second or greater.
 4. A methodof sorting cells, comprising screening cellular material to identifycells with a desired phenotype, wherein the cellular material isscreened at a rate of 50,000 cells per second or greater; and whereinsaid cellular material is screened in an array with through-holes. 5.The method of claim 4, wherein said cells of said desired phenotype areextracted from said array.
 6. The method of claim 5, wherein saidextracting comprises releasing said cells of said desired phenotypeusing electromagnetic radiation.
 7. The method of claim 6, wherein eachthrough-hole of said array comprises from 0 to about 5 cells and atleast 30% of the through-holes of the array comprise at least one cell.8. The method of claim 1, wherein the cellular material is obtained froma human subject and/or comprises less than 5% of HSCs and HSPCs.
 9. Themethod of claim 2, wherein greater than 95% of the extracted cells areHSCs and/or HSPCs.
 10. The method of claim 2, wherein greater that 95%of the extracted cells are cells of said desired phenotype.
 11. Themethod of claim 2, wherein greater than 95% of the extracted cells areviable as determined by evaluating the cells within 5 hours from saidextracting.
 12. The method of claim 2, wherein the extracted cells aresuitable for therapeutic use without the need for additionalsterilization steps.
 13. The method of claim 2, wherein the extractedcells are essentially free of pathogens.
 14. The method of claim 2,wherein the extracted cells comprise less than 0.1% of pathogens. 15.The extracted cells obtained by the method of claim
 2. 16. Apharmaceutical composition comprising the extracted cells of claim 15.